Compositions and methods for detecting ccr2 receptors

ABSTRACT

Among the various aspects of the present disclosure is the provision of compositions of imaging agents and methods for use in detecting, monitoring, and evaluating CCR2 associated diseases, disorders, and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/344,677 filed on 2 Jun. 2016, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberHHSN268201000046C and HL131908 awarded by National Institutes of Health.The government has certain rights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form comprising nucleotide and/or aminoacid sequences of the present invention. The subject matter of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to compositions of imagingagents and methods of making and detection thereof.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofcompositions of imaging agents for use in detecting, monitoring, andevaluating CCR2 associated diseases, disorders, and conditions.

Briefly, therefore, the present disclosure is directed to compositionsand methods to detect, monitor, and evaluate conditions associated withCCR2 upregulation or overexpression.

The present disclosure provides for compositions for imaging agents.

The present disclosure provides for pharmaceutical compositioncomprising the imaging agents.

The present disclosure provides for methods for imaging inflammationassociated with diseases, disorders, and conditions associated withCCR2.

The present disclosure provides for imaging agents comprising a CCR2binding peptide; a radiolabel; and a nanoparticle, a chelator, or alinker.

In some embodiments, the imaging agent further comprises a linker.

In some embodiments, the CCR2 binding peptide comprises a linear ECL1peptide or a cyclized ECL1i peptide; an amino acid sequenceThr-Phe-Leu-Lys (SEQ ID NO: 17); SEQ ID NO: 17 comprising one or morechemical modifications that confer resistance to proteolysis; SEQ ID NO:17 comprising one or more conservative substitutions;Thr-Phe-Leu-Lys-Cys (SEQ ID NO: 1); SEQ ID NO: 1 comprising one or morechemical modifications that confer resistance to proteolysis; SEQ ID NO:1 comprising one or more conservative substitutions; X1-TFLKC-X2 (SEQ IDNO: 2), wherein X1 is absent, is glycine, or represents an amino acidsequence selected from the group consisting of AG, LG, YLG, and HYLG;and X2 independently is absent, is methionine, or represents an aminoacid sequence selected from the group consisting of MA, MAN, MANG,MANGF, MANGFV, MANGFVW, MANGFVWE, and MANGFVWEN; SEQ ID NO: 2 comprisingone or more chemical modifications that confer resistance toproteolysis; SEQ ID NO: 2 comprising one or more conservativesubstitutions; X1-TFLK-X3 (SEQ ID NO: 18), wherein X1 is absent, isglycine, or represents an amino acid sequence selected from the groupconsisting of AG, LG, YLG, and HYLG; and X3 independently is absent oris alanine; SEQ ID NO: 18 comprising one or more chemical modificationsthat confer resistance to proteolysis; or SEQ ID NO: 18 comprising oneor more conservative substitutions.

In some embodiments, the CCR2 binding peptide comprises amino acids andall or a portion of the amino acids are in L configuration or in Dconfiguration; the imaging agent is stored in a physiological pH,optionally, at about pH of 7.4; the CCR2 binding peptide is covalentlylinked to the nanoparticle; or the CCR2 binding peptide is no more than18 amino acids in length.

In some embodiments, the CCR2 binding peptide is selected from the groupconsisting of: LGTFLKC (SEQ ID NO: 3); HYLGTFLKCMA (SEQ ID NO: 4);LGTFLKCMA (SEQ ID NO: 5); HYLGTFLKC (SEQ ID NO: 6); GTFLKCMANGF (SEQ IDNO: 7); TFLKCMANGFV (SEQ ID NO: 8); HYLGTFLKCMANGFVWEN (SEQ ID NO: 9);LGTFLK (SEQ ID NO: 19); AGTFLKC (SEQ ID NO: 20); LGTFLKA (SEQ ID NO:21); GTFLK (SEQ ID NO: 22); AGTFLKA (SEQ ID NO: 23); a sequence derivingfrom any of SEQ ID NO: 3 to 10, or 19 to 23 by one or more chemicalmodifications that confer resistance to proteolysis; or a sequencederiving from any of SEQ ID NO: 3 to 9 or 19 to 23 by one or moreconservative substitutions.

In some embodiments, the CCR2 binding peptide consists of LGTFLKC (SEQID NO: 3).

In some embodiments, the radiolabel comprises ²H (D or deuterium), ³H (Tor tritium), ¹¹C, ¹³C, ¹⁴O, ⁶⁴CU, ⁶⁷CU, ¹⁷⁷Lu, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O,¹⁸F, ⁸⁹Sr, ³⁵S, ¹⁵³Sm, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Tl ^(99m)Tc, ⁹⁰Y, or⁸⁹Zr; the radiolabel comprises oxygen-15 water, nitrogen-13 ammonia,[⁸²Rb] rubidium-82 chloride, [¹¹C], [¹¹C] 25B-NBOMe, [¹⁸F] Altanserin,[¹¹C] Carfentanil, [¹¹C] DASB, [¹¹C] DTBZ, [¹⁸F]Fluoropropyl-DTBZ, [¹¹C]ME@HAPTHI, [¹⁸F] Fallypride, [¹⁸F] Florbetaben, [¹⁸F] Flubatine, [¹⁸F]Fluspidine, [¹⁸F] Florbetapir, [¹⁸F] or [¹¹C] Flumazenil, [¹⁸F]Flutemetamol, [¹⁸F] Fluorodopa, [¹⁸F] Desmethoxyfallypride, [¹⁸F]Mefway, [¹⁸F] MPPF, [¹⁸F] Nifene, Pittsburgh compound B, [¹¹C]Raclopride, [¹⁸F] Setoperone, [¹⁸F] or [¹¹C] N-Methylspiperone, [¹¹C]Verapamil, [¹¹C] Martinostat, Fludeoxyglucose (¹⁸F)(FDG)-glucoseanalogue, [¹¹C] Acetate, [¹¹C] Methionine, [¹¹C] Choline, [¹⁸F]Fluciclovine, [¹⁸F] Fluorocholine, [¹⁸F] FET, [¹⁸F] FMISO, [¹⁸F]3′-fluoro-3′-deoxythymidine, [⁶⁸Ga] DOTA-pseudopeptides, [⁶⁸Ga] PSMA, or[¹⁸F] Fluorodeoxysorbitol (FDS); the chelator comprises NHS-MAG3, MAG3,DTPA, 3p-C-NE3TA, 3p-C-NOTA, 3p-C-DE4TA, ATSM, tetraazamacrocyclicligands (e.g., DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), DOTA-NHS, pSCN-Bn-DOTA, pNH₂-Bn-DOTA, TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid,TETA-octreotide (OC)), hexaazamacrobicyclic cage-type ligands (e.g.,Sarcophogine chelators), cross-bridged tetraamine ligands (e.g., CB-TE2A(4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane)),6-Hydrazinopridine-3-carboxylic acid (Hynic), NHS-Hynic,2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA); or the nanoparticle comprises ananocluster or nanostructure; organic, inorganic, or lipidnanostructures; the nanoparticle comprises iron oxide, gold, goldnanoclusters (AuNC), gold nanorods (AuNR), copper (Cu), quantum dots,carbon nanotubes, carbon nanohorn, gadolinium (Gd), dendrimers,dendrons, polyelectrolyte complex (PEC) nanoparticles, calcium phosphatenanoparticles, perfluorocarbon nanoparticles (PFCNPs), lipid-basednanoparticles, liposomes, or micelles; or the linker comprises achemical or physical bond; PEG, TA-PEG-Maleimide, TA-PEG-OMe, TA-PEG, anisothiocyanate group, a carboxylic acid or carboxylate groups, adendrimer, a dendron, Fmoc-protected-2,3-diaminopropanoic acid, ascorbicacid, a silane linker, minopropyltrimethoxysilane (APTMS), dopamine, 2thiol groups, 2 primary amines, a carboxylic acid and primary amine,maleimide and thiol, hydrazide and aldehyde, or a primary amine andaldehyde, an amide, a thioether, a disulfide, an acetyl-hydrazone group,a polycyclic group, a click chemistry (CC) group.

In some embodiments, the chelator is conjugated to the CCR2 bindingpeptide and the chelator is radiolabeled; or the CCR2 binding peptide isconjugated to a nanoparticle.

In some embodiments, the radiolabel is ⁶⁴Cu.

In some embodiments, the CCR2 binding peptide is conjugated to ananoparticle comprising a gold nanocluster.

In some embodiments, the gold nanocluster is loaded with a radiolabel.

In some embodiments, the chelator comprises a tetraazamacrocyclicligand; DOTA; TETA; or2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA).

In some embodiments, the chelator is conjugated to a cysteine residue ofthe CCR2 binding peptide.

In some embodiments, the imaging agent comprises: a ⁶⁴Cu-DOTA-ECL1i PETor SPECT imaging agent; an ECL1i peptide conjugated to a goldnanocluster, wherein the gold nanocluster is loaded with ⁶⁴CU;⁶⁴Cu-DOTA-ECL1i; ⁶⁴CuAuNCs-ECL1i; a multivalent imaging agent(optionally, ⁶⁴CuAuNCs-ECL1i), exhibiting extended pharmacokinetics forlong-term CCR2 receptor detection and targeted theranostics; or amonovalent (optionally, ⁶⁴Cu-DOTA-ECL1i), exhibiting fastpharmacokinetics for efficiently for rapid or serial imaging of CCR2receptors.

In some embodiments, the imaging agent is a PET imaging agent; is aSPECT imaging agent; targets CCR2 receptors; detects CCR2 receptorup-regulation; or detects elevated CCR2 expression.

Another aspect of the present disclosure provides for a method ofdetecting a CCR2 receptor comprising administering to a subject animaging agent or detecting the imaging agent.

In some embodiments, the detecting of the CCR2 receptor comprisespositron emission tomography (PET) imaging, and single photon emissioncomputed tomography (SPECT) imaging, mass spectrometry, gamma imaging,magnetic resonance imaging (MRI), magnetic resonance spectroscopy,fluorescence spectroscopy, CT, ultrasound, or X-ray.

In some embodiments, the method detects a CCR2 associated disease,disorder, or condition is selected from the group consisting of:atherosclerosis; abdominal aortic aneurysm; acquired metabolic disease;acute cystitis; acute lung injury; acute proliferativeglomerulonephritis; acute or chronic sinusitis; age-related maculardegeneration; alcoholic hepatitis; allergic asthma; allergicconjunctivitis; allergic rhinitis; alveolitis; angiostenosis;anthracosis; ariboflavinosis; arteriosclerosis; artery disease;arthritis; asthma; atherogenesis; atheroma; atherosclerosis; atopicdermatitis; autoimmune disease; autoinflammation; bacterial infection;bacteriuria; bladder cancer; bone cancer; bone inflammation disease;brain trauma; breast cancer; bronchiolitis; bronchiolitis obliteranssyndrome; cancer; cardiac infarction; cardiovascular disease; carotidartery disease; CCR2 associated neurological disorders; Cd3zetadeficiency; central nervous system disease; cerebral aneurysms; cervicalcancer; chagas disease; chorioamnionitis; chronic heart failure; chroniclung disease; chronic lymphocytic leukemia; chronic myelocytic leukemia;chronic obstructive pulmonary disease (COPD); chronic respiratory viralinfection; chronic urticaria; colitis; colon cancer; complex regionalpain syndrome; coronary artery aneurysm; crescentic glomerulonephritis;Crohn's Disease; cystitis; cytomegalovirus retinitis; degeneration ofmacula and posterior pole; demyelinating disease; dengue shock syndrome;denture stomatitis; dermatosis syndrome; diabetes; diabetes mellitus,noninsulin-dependent; diabetic angiopathy; diabetic complications;diabetic macular edema; diabetic microangiopathy; diabetic nephropathy;diabetic retinitis; diabetic retinopathy; diastolic cardiomyopathies;encephalitis; endocervicitis; endometrial stromal sarcoma;endometriosis; Erdheim-Chester disease; extrapulmonary tuberculosis;extrinsic cardiomyopathy; eye disease; fibroid lung; fungal pneumonia;gingivitis; glomerulonephritis; gum disease; Hamman-Rich syndrome; headand neck cancer, herpes simplex virus keratitis; HIV-1; Hodgkin'sdisease; hyperhomocysteinemia; idiopathic anterior uveitis; idiopathicinterstitial pneumonia; idiopathic pulmonary fibrosis; inflammationafter cataract surgery; inflammatory bowel diseases; inflammatorydisease; influenza; interstitial lung disease; invasive staphyloccocia;ischemia of lower members of the heart; ischemia-reperfusion injury;Israeli tick typhus; Kawasaki disease; keratitis; kidney disease;leptospirosis; limb ischemia; lipid pneumonia; lipodystrophy; lipoidnephrosis; lung cancer; lung disease; lung injury; lung transplantation;macular degeneration, age-related, 1; macular holes; malaria; malignantmyeloma; mast-cell leukemia; meningitis; mesangial proliferativeglomerulonephritis; metabolic disease; microvascular complications ofdiabetes 1; monocytic leukemia; multiple myeloma; multiple sclerosis;mycobacterium tuberculosis; myocardial infarction; myocarditis;necrosis; neovascular inflammatory disease; nephritis; nephrosclerosis;neural tube defects; neuritis; neuroinflammation; nonspecificinterstitial pneumonia; obesity; ophthalmic disorder; organ allograftrejection; overnutrition; pain; pain from a sciatic nerve; papillaryconjunctivitis; pelvic inflammatory disease; periodonitis; periodontaldiseases; periodontitis; peripheral artery disease; peripheral pain;peritonitis; pleural tuberculosis; pleurisy; pneumoconiosis; pneumonia;post-thrombotic syndrome; primary graft dysfunction (PGD) (a reperfusioninjury after transplant); proliferative glomerulonephritis; prostatecancer; psoriasis; psoriatic arthritis; pulmonary alveolar proteinosis;pulmonary fibrosis; pulmonary sarcoidosis; purulent labyrinthitis;pyelonephritis; radiculopathy; renal fibrosis; renal insufficiency;reperfusion disorders; respiratory system disease; restenosis; retinaldegeneration; retinal vascular occlusion; retinal vasculitis; retinalvein occlusion; rheumatoid arthritis; rhinoscleroma; sarcoidosis;sarcoidosis 1; scleritis; secondary progressive multiple sclerosis;severe acute respiratory syndrome; silicosis; solid tumor; stachybotryschartarum; stomach cancer; stromal keratitis; systemic lupuserythematosus; transient cerebral ischemia; transplant arteriosclerosis;trypanosomiasis; tuberculosis; tuberculous meningitis; type II diabetes;ulcerative colitis; ureteral disease; urinary system disease; urinarytract obstruction; uveitis; vangl1-related neural tube defect; vasculardisease; vascular permeability and attraction of immune cells duringmetastasis; vasculitis; verruciform xanthoma of skin; viral infection;viral encephalitis; viral meningitis; vitreoretinopathy; orxanthogranulomatous pyelonephritis; acute lung injury; inflammation;primary graft dysfunction (PGD); asthma; pulmonary fibrosis; COPD;atherosclerosis; lung transplant; lung injury; COPD; atherosclerosis;cancer; prostate cancer; organ transplant; metabolic disease, type IIdiabetes; multiple sclerosis; rheumatoid arthritis; pain; pulmonaryfibrosis; or reperfusion in a lung transplant; inflammation associatedwith a CCR2 associated disease, disorder, or condition; or inflammationassociated with lung injury, graft transplantation, atherosclerosis,tumor cells, or cancer.

In some embodiments, the method further comprises evaluating ormonitoring a CCR2 associated disease, disorder, or condition; oradministering a CCR2 antagonist to a subject.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is a structure of DOTA-ECL1i (synthetic methods are described inExamples 1, 2, and 3).

FIG. 2A-FIG. 2B is a series of plots showing the characterization of⁶⁴Cu-DOTA-ECL1i. (FIG. 2A) HPLC profiles of DOTA-ECL1i (UV, black) and⁶⁴Cu-DOTA-ECL1i (radioactivity, green); (FIG. 2B) Mass spectrometryspectrum of Cu-DOTA-CCR2 (arrow).

FIG. 3A-FIG. 3C is a series of graphs and histograms showing lungtransplant recipient CCR2 expression promotes monocyte recruitment intolung grafts and mediates ischemia reperfusion injury. (FIG. 3A) ArterialpaO₂ levels 6 h after transplantation of B6 wild type (WT) lungs into B6wild type (n=4) or B6 CCR2-deficient (CCR2^(−/−)) (n=4) recipients.**p<0.05. Contour plots depicting recipient (CD45.2) versus donor(CD45.1) hematopoietic cells in B6 CD45.1 lung grafts 6 hours aftertransplantation into (FIG. 3B) B6 CD45.2 wild type or (FIG. 3C) B6CD45.2 CCR2-deficient hosts. Monocytes (CD11b⁺Ly6C^(hi)) are gated onrecipient (CD45.2) (top panels) or donor (CD45.1) (bottom) hematopoieticcells. Numbers are percentages in respective gates. Histograms depictCCR2 expression on recipient (CD45.2) (top) or donor (CD45.1) (bottom)monocytes. Solid line: CCR2; shaded: isotype control). Plots in (FIG.3A) and (FIG. 3B) are representative of at least 3 independentexperiments in each group. Lung grafts were stored in low potassiumdextran solution at 4° C. for 18 hours prior to transplantation.

FIG. 4 is a bar graph showing CCR2 RT-PCR confirmed the up-regulation ofCCR2 following lung transplantation. RT-PCR showing the relativeexpression of CCR2 mRNA in native and donor lungs 24 hours after B6 wildtype→B6 wild type transplantation (n=4).

FIG. 5A-FIG. 5C is a series of chemical structures, an image and ahistogram showing the synthetic scheme and characterization of⁶⁴CuAuNCs-ECL1i showed straightforward preparation and uniform sizedistribution. (FIG. 5A) Synthetic scheme of ⁶⁴CuAuNCs-ECL1i. (FIG. 5B)Transmission electron microscopy and (FIG. 5C) dynamic light scatteringof decayed CuAuNCs-ECL1i.

FIG. 6A-FIG. 6B is a series of bar graphs showing biodistributionstudies in B6 wild type mice demonstrate different pharmacokineticsbetween ⁶⁴Cu-DOTA-ECL1i and ⁶⁴CuAuNCs-ECL1i. (FIG. 6A) ⁶⁴Cu-DOTA-ECL1ishowed rapid renal clearance at 1 hour and (FIG. 6B) ⁶⁴CuAuNCs-ECL1ishowed extended pharmacokinetics at 1, 4, and 24 hours (n=4/group).

FIG. 7A-FIG. 7B is a PET image and a bar graph showing ¹⁵O-water PETimaging demonstrated comparable blood flow between donor and nativelungs after transplantation. (FIG. 7A) Representative ¹⁵O-water PETimage at 1 hour following B6 wild type→B6 wild type lung transplantationshowing comparable signals in both lungs. The PET imaging was performedas a 0-10 dynamic scan immediately after the injection of ¹⁵O-water.(FIG. 7B) Quantitative uptake of ¹⁵O-water in native lungs and donorgrafts showing comparable signals (n=4).

FIG. 8A-FIG. 8B is a series of PET images and bar graphs showing CCR2 isdetected by ⁶⁴CuDOTA-ECL1i using PET. (FIG. 8A) Representative⁶⁴CuDOTA-ECL1i PET/CT images in B6 wild type→B6 wild type and B6 wildtype→B6 CCR2-deficient lung transplant combinations at 1, 4 and 24 hoursafter transplantation. Tracer uptake was observed in the donor lungs inboth models. Quantitative uptake analysis in lung grafts and nativelungs after (FIG. 8B) B6 wild type→B6 CCR2-deficient and (FIG. 8C) B6wild type→B6 wild type pulmonary transplantation. L: liver, K: kidney,B: bladder. Circle: donor lung (n=4/group).

FIG. 9 is a PET image of ⁶⁴Cu-DOTA-ECL1i PET imaging in naïve mousedemonstrated imaging specificity.

FIG. 10A-FIG. 10B is a PET image and bar graphs showing competitive CCR2receptor blocking study confirmed ⁶⁴Cu-DOTA-ECL1i imaging specificity.(FIG. 10A) Representative ⁶⁴Cu-DOTA-ECL1i PET blocking image during a0-60 min dynamic scan. PET imaging was performed at 1 hour after B6 wildtype→B6 wild type lung transplantation with the co-injection ofnon-radiolabeled ECL1i and ⁶⁴Cu-DOTA-ECL1i at 1000:1 molar ratio. Circleoutlines uptake in donor graft. (FIG. 10B) Quantitative uptake of⁶⁴Cu-DOTA-ECL1i in native lungs and donor grafts showing comparableaccumulation. (FIG. 10C) Bar graph shows donor graft/native lung SUVuptake ratio (n=4).

FIG. 11A-FIG. 11B is a PET image and a bar graph showing ¹⁸F-FDG PETimaging of uptake in the donor lung. (FIG. 11A) Representative ¹⁸F-FDGPET image obtained at 1 hour after lung transplantation showing uptakein the donor lung after B6 wild type→B6 wild type lung transplantation.(FIG. 11B) Quantitative uptake of ¹⁸F-FDG in native lungs and donorgrafts (n=4).

FIG. 12A-FIG. 12D is a series of PET images and bar graphs showing CCR2is detected by ⁶⁴CuAuNCs-ECL1i with enhanced efficiency using PET. (FIG.12A) Representative ⁶⁴CuAuNCs-ECL1i and ⁶⁴CuAuNCs nanoclusters PET/CTimages in B6 wild type→B6 wild type lung transplantation model at 24hours following engraftment. The targeted nanocluster showed significantuptake in the donor lung while the non-targeted counterpart showedminimum non-specific retention. Quantitative uptake analysis of (FIG.12B) ⁶⁴CuAuNCs and (FIG. 12C) ⁶⁴CuAuNCs-ECL1i in lung grafts and nativelungs after B6 wild type→B6 wild type pulmonary transplantation and(FIG. 12D) Donor graft/native lung uptake ratios for ⁶⁴CuAuNCs-ECL1i and⁶⁴CuAuNCs PET after B6 wild type→B6 wild type pulmonary transplantation.Circle: donor lung (n=4/group).

FIG. 13A-FIG. 13C is a series of PET images and bar graphs showing⁶⁴Cu-DOTA-vMIP-II PET imaging in LPS injured mouse lung. (FIG. 13A) PETimages of mice post intratracheal PBS or LPS, injected with i.v.⁶⁴Cu-DOTA-vMIP-II for PET at 3-144 h later. White arrow indicates thevMIP-II lung signal. (FIG. 13B) Standardized uptake value (SUV) of lungimages. n=3-8 mice/time point. (FIG. 13C) Mean fluorescent intensity(MFI) of cell types from lung digests co-localizedDOTA-vMIP-II-Dylight550 by flow cytometry. Mice were injected withDOTA-vMIP-II-Dylight550 at 24 or 48 h post PBS or LPS. n=4 experiments;*p<0.05 compared to PBS.

FIG. 14A-FIG. 14G is a series of images and bar graphs showing PETimaging CCR2 in LPS-injured mouse lung. Mice post intratracheal PBS orLPS. (FIG. 14A, FIG. 14B) Immunostaining for CCR2. (FIG. 14C, FIG. 14D)PET/CT images of mice injected with i.v. ⁶⁴Cu-DOTA-ECL1i at 24 h. Whitearrow indicates lung signal. (FIG. 14E) Quantitation of lung images at24-144 h post-LPS. (FIG. 14F) Dose-response (20-fold) and non-PBScontrols, naïve and CCR2 null mice. Blocked were mice treated withexcess cold tracer. n=3-8 mice/time point. (FIG. 14G) Biodistribution at24 h. *p<0.05 LPS compared to PBS treatment.

FIG. 15 is a bar graph showing ECL1i Dylight550⁺ cells in vivo. Micetreated with intratracheal PBS or LPS were given i.v.fluorescent-labeled ECL1i at 24 h. Lung cell digests were analyzed byflow cytometry for ECL1i-Dylight550⁺ cells. Shown are mean±SD of 3independent experiments. *p<0.05.

FIG. 16A-FIG. 16D is a series of images and plots showing CCR2 detectionin lung tissue from subjects with COPD. (FIG. 16A) Immunostaining forCCR2, bar, 50 μm B. Quantitation of A, p<0.05. (FIG. 16C)Autoradiography of ⁶⁴Cu-DOTA-ECL1i binding of lung tissues sections onglass slides. (FIG. 16D) Flow cytometry of CD45⁺ ECL1i-647⁺ cells from adigested COPD lung sample.

FIG. 17 is a flow chart showing the GMP preparation of ⁶⁴Cu-DOTA-ECL1i.

FIG. 18 is a flow chart showing the Phase 0/Early Phase 1 safety anddosimetry study.

FIG. 19A-FIG. 19C is a series of flow charts describing the study designof CCR2 imaging using the ECL1i peptide. (FIG. 19A) Peptide labeling andstability. (FIG. 19B) Mouse imaging studies. The number of mice in eachtreatment group is indicated. Bold boxes indicate in vivo PET/CT imagingstudies. Time-activity analysis was performed on a subgroup of PETimages obtained at 24 h post-treatment. (FIG. 19C) Human lung tissuesstudies. A subgroup of lung tissues from subjects with COPD displayingelevated levels of CCR2 positive cells was analyzed for ⁶⁴Cu-DOTA-ECL1ibinding using autoradiography followed by blocking (n=6).

FIG. 20A-FIG. 20B is a series of spectra characterizing ⁶⁴Cu-DOTA-ECL1i.(FIG. 20A) Mass spectrometry of DOTA-ECL1i (arrow). (FIG. 20B)DOTA-ECL1i was labeled with ⁶⁴Cu and assayed for chemical purity andradiochemical purity determined by HPLC. Shown are profiles ofDOTA-ECL1i (UV, black) and ⁶⁴Cu-DOTA-ECL1i (radioactivity, green).

FIG. 21 is an HPLC spectrum showing in vivo stability of⁶⁴Cu-DOTA-ECL1i. The radiolabeled probe ⁶⁴Cu-DOTA-ECL1i was analyzed byHPLC to determine the retention time. Shown are representative traces of⁶⁴Cu-DOTA-ECL1i as a standard (black), and samples obtained from naive,wild type mice injected intravenously with the radioprobe. After one h,plasma (blue) and the lung homogenate (red) was obtained and assayed byHPLC. Data are representative of n=3.

FIG. 22A-FIG. 22B is a series of images and a bar graph showing thebiodistribution of ⁶⁴Cu-DOTA-ECL1i in mice with lung injury. Mice werenot treated (naive), or administered intratracheal PBS or LPS. (FIG.22A) Immunostaining for CCR2, 24 h after intratracheal PBS or LPSdelivery. Tissues were counterstained with hematoxylin. Images werecaptured at 400× magnification, bar=50 μm. (FIG. 22B) Biodistribution of⁶⁴Cu-DOTA-ECL1i at 24 h, compared to the PBS and naive groups. Shown isthe mean±SD, n=4/group, *P<0.001 by two-way ANOVA with Tukey's test.

FIG. 23A-FIG. 23D is a series of images, a plot and bar graphs depictingPET imaging data of ⁶⁴Cu-DOTA-ECL1i in mouse lung injury model. Micewere administered intratracheal PBS or LPS, followed by injection withi.v. ⁶⁴Cu-DOTA-ECL1i. FIG. 23A. Representative PET images of maximumintensity projections (MIP) reconstructed PET/CT (center) and fromindicated planes acquired at 24 h post-treatment and after injectionwith ⁶⁴Cu-DOTA-ECL1i. White arrows point to labeled organs. (FIG. 23B)Time activity curves of ⁶⁴Cu-DOTA-ECL1i in heart and lungs 0-60 min,n=3/group from a subgroup described in FIG. 23C. *P<0.001 by unpaired,two-tailed t-test comparing lung PBS and LPS groups. (FIG. 23C)⁶⁴Cu-DOTA-ECL1i lung uptake acquired at indicated time post-treatment(PBS, n=5; LPS n=7 at 24 h, n=3 at 48 h, n=3 at 144 h). (FIG. 23D)Uptake in lung after delivery of low (n=3), intermediate (n=7) and highdose (n=3) LPS. The intermediate dose activity is the same data as shownin C for comparison. Data in FIG. 23B, FIG. 23C, FIG. 23D are themean±SD. *P<0.001 compared to PBS in FIG. 23C and *P<0.001 and **P=0.006compared to low dose LPS in FIG. 23D, by one-way ANOVA with Tukey's test

FIG. 24A-FIG. 24B is a PET image and a bar graph depicting thespecificity of ⁶⁴Cu-DOTA-ECL1i imaging in LPS lung injury model. Micewere not treated (naive), or delivered intratracheal LPS and 24 h laterinjected with ⁶⁴Cu-DOTA-ECL1i for PET/CT imaging. (FIG. 24A)Representative transverse PET images of mouse lung obtained from theindicated experimental condition: not treated WT mice (naive n=4);LPS-treated WT mice (WT, n=7; from FIG. 2B); LPS-treated WT co-injectedwith non-radiolabeled ECL1i (blocked, n=4) or LPS-treated CCR2-deficientmice (CCR2^(−/−), n=4). (FIG. 24B) Quantification of lung activity afterthe treatment described in A. Data are the mean±SD. *P<0.001 compared toLPS-treated WT mice injected with ⁶⁴Cu-DOTA-ECL1i, by one-way ANOVA withTukey's test.

FIG. 25 is a bar graph showing the detection of ECL1i-Dylight 550 inlung immune cells after LPS-induced lung injury. Mice treated withintratracheal PBS or LPS were given intravenous fluorescent-labeledECL1i, or non-injected at 24 h. One h later, lung cell digests wereanalyzed by flow cytometry for ECL1i-Dylight550⁺ cells using cell-typespecific antibodies. Cell types: PMN, neutrophils; Mono, monocytes;Macro, macrophages; DC, dendritic; T and B cells, lymphocytes. Shown aremean±SE of 4 independent experiments, total n=4/PBS group and n=5/LPSgroup injected with ECL1i; non-injected control mice for flow cytometry,n=4/PBS group, n=4 LPS/group. *P=0.001 compared to PBS treatment byunpaired t-test.

FIG. 26A-FIG. 26D is a series of images and plots showing⁶⁴Cu-DOTA-ECL1i binding in human lung samples from subjects with severeCOPD. (FIG. 26A) Photomicrograph of CCR2 immunostaining in lung tissuefrom non-COPD lung donors, and a subject with severe COPD demonstratinga high number of CCR2-expressing cells. Captured at 100× magnification;bar=100 μm; CCR2, red; DAPI, blue. (FIG. 26B) Quantification of CCR2staining area relative to DAPI in lung sections from donor (n=11) andCOPD (n=16) subjects, from photographs acquired at 200×. (FIG. 26C)Representative autoradiography of ⁶⁴Cu-DOTA-ECL1i binding to lungtissues sections on glass slides. (FIG. 26D) Quantification ofautoradiography of lung sections from subjects with COPD. The counts ineach blocked sample were compared to those non-blocked tissues, whichwere set as 1.0, n=6. Bars in B and D describe the median value, and aresignificantly different between groups, *P=0.002 in B and D by theMann-Whitney test for non-parametric data.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thata CCR2 binding peptide adapted as a PET probe can detect CCR2 receptorsor inflammation in a mouse model and human tissues.

As described herein, a ⁶⁴Cu-radiolabeled ECL1i peptide radiotracer(⁶⁴Cu-DOTA-ECL1i) and ECL1i-conjugated gold nanoclusters doped with ⁶⁴Cu(⁶⁴CuAuNCs-ECL1i) showed specific detection of CCR2. Due to its fastpharmacokinetics ⁶⁴Cu-DOTA-ECL1i functioned efficiently for rapid andserial imaging of CCR2. The multivalent ⁶⁴CuAuNCs-ECL1i with extendedpharmacokinetics is favored for long-term CCR2 detection and potentialtargeted theranostics.

As described herein, the developed imaging agents (e.g., PET tracer of⁶⁴Cu-DOTA-ECL1i) have shown the specific and sensitive detection of CCR2receptor up-regulated at inflammatory sites in multiple animal models.The biological characterization and confirmation of CCR2 receptor inthese models show support for the robust PET imaging agent disclosedherein for clinical use.

As described herein, CCR2 binding peptide, ECL1i, adapted as a positronemission tomography (PET) radiotracer can be used to detect a 3.5-foldgreater activity in the lungs of mice injured after intratrachealdelivery of lipopolysaccharide compared to the saline control group(P<0.001).

As described herein, levels of CCR2⁺ cells varied in human lung tissueamong subjects with chronic obstructive lung disease (range 1.62 to24.11 percent cells per tissue sample).

As described herein, the radiotracer ⁶⁴Cu-DOTA-ECL1i can be used todetect inflammation in human lung tissue from subjects with COPD usingautoradiography (specificity determined by non-radioactive blockade,54.5%: P=0.002), suggesting clinical application if approved for in vivohuman use.

Also described herein are detailed methods for mouse studies (see e.g.,Example 4); hCCR2 binding assay (see e.g., Example 5); lung tissuebinding (see e.g., Example 5); toxicology and dosimetry (see e.g.,Example 6A); manufacturing and controls (CMC) and standard operatingprocedures (SOPs) for ⁶⁴Cu-DOTA-ECL1i production (see e.g., Example 6B);and methods for phase 0 and early phase 1 clinical trials (see e.g.,Example 6C).

Imaging Agent/CCR Molecular Probes

In these studies, the use of CCR2-specific molecular probes or imagingagents are reported.

As described herein, a novel imaging agent was developed through theconjugation of a novel targeting peptide for positron emissiontomography (PET) imaging in mouse models of CCR2 associated diseases,disorders, and conditions (e.g., lung injury using lipopolysaccachride(LPS), transplant-mediated ischemia reperfusion injury, atherosclerosis,prostate cancer).

The presently disclosed work provides a powerful tool for the sensitiveand specific detection of CCR2 to track inflammatory monocytes invarious pathological conditions and lay the foundation for newapproaches for the diagnosis and treatment of immune-mediated processes(e.g., ischemia reperfusion injury and rejection in transplantrecipients) using imaging modalities (e.g., PET).

As described herein, an imaging agent can comprise a radiolabel, a CCR2binding peptide, a nanoparticle, and/or a chelator. The imaging agentcan further comprise a linker group. An imaging agent can be formulatedto be detected by any method of imaging known in the art. For example,the imaging agent can be detected using a PET scanner (i.e., the imagingagent is a PET imaging agent or tracer). As another example, the imagingmodality used to detect the imaging agent can comprise PET, SPECT, massspectrometry, MRI, NMR, fluorescence spectroscopy, computed tomography(CT), ultrasound, or X-ray.

The present disclosure provides the first known imaging probe availablefor CCR2 detection. Currently, there are no known available methods forthe non-invasive imaging of CCR2. To fill this gap, the presentdisclosure provides for CCR2 receptor imaging agents (e.g., aradiolabeled positron emission tomography (PET) tracer) have beendeveloped based on the ECL1i peptide(DLeu-Gly-DThr-DPhe-DLeu-DLys-DCys).

Provided herein are positron emission tomography (PET) and single photonemission computed tomography (SPECT) imaging probes using a CCR2 bindingpeptide (e.g., the ECL1 (C) inverso peptide (LGTFLKC) to image theup-regulation of chemokine receptor CCR2 in both in vitro cell studiesand a range of animal disease models. The peptide can be conjugated withmacrocyclic chelator for radiolabeling with various radionuclides orgrafted on a nanostructure with controlled physicochemical properties.The conjugation strategy and compositions of developed imaging probeshave been optimized for enhanced binding affinity and improved in vivopharmacokinetics.

PET imaging has been performed in animal disease models tonon-invasively track the specific cell population expressing CCR2receptor at site-of-interest. In other studies, fluorescent or othertags can be conjugated on the peptide to track the probe fornon-radioactive studies.

For example, the present disclosure describes a ⁶⁴Cu-DOTA-ECL1i tracerdeveloped for PET imaging of CCR2 receptor up-regulation associated withinflammation in multiple models of disease including a murine lungtransplant model, a murine lung lipopolysaccharide (LPS) injury model,an apolipoprotein E-deficient (ApoE^(−/−)) mouse atherosclerosis model,and a prostate cancer xenograft mouse model.

As described herein, the CCR2 imaging specificity and sensitivity havebeen well characterized in pre-clinical studies.

Lung Transplantation Model.

The CCR2 targeting specificity has been confirmed by using CCR2knock-out (CCR2^(−/−)) mice in the lung transplantation model.

Specifically, strong PET signal was observed in the donor lung of wildtype to wild type mouse lung transplantation model. In the wild type toCCR2^(−/−) lung transplantation model, PET signal was only detected inthe donor lung, not the recipient lung, which demonstrated the CCR2targeting specificity of ⁶⁴Cu-DOTA-ECL1i. The characterization of CCR2receptor was confirmed using flow cytometry and immunohistochemistry.The CCR2 imaging specificity was also verified by performing acompetitive receptor blocking study through the co-injection of⁶⁴Cu-DOTA-ECL1i and excess amount of ECL1i peptide (ECL1i vs.⁶⁴Cu-DOTA-ECL1i molar ratio=500:1) in the wild type to wild type mouselung transplantation model. Quantitative PET data analysis showedsignificantly blocked signal at donor lung compared to uptake in thedonor lung without the blocking agent, indicating the CCR2 specifictracer uptake.

LPS Injury Model.

The CCR2 targeting specificity has been confirmed by using CCR2knock-out (CCR2^(−/−)) mice in the lung LPS injury model.

The imaging sensitivity of ⁶⁴Cu-DOTA-ECL1i was characterized in the lungLPS injury model. Specifically, following the administration of LPS atlow, intermediate and high dose level to generate different levels ofinjury, ⁶⁴Cu-DOTA-ECL1i PET imaging was performed at 24 h post injuryfollowing the same imaging protocol. The PET signal intensities in thelungs correlated with the injury level, which demonstrated thesensitivity of this PET tracer detecting inflammation.

Atherosclerosis Model.

In mouse apoE^(−/−) atherosclerosis model, the ⁶⁴Cu-DOTA-ECL1i tracerclearly showed the detection of plaque at aortic arch and greatpotential to track the progression of disease.

Prostate Cancer Model.

In a mouse PC3 prostate cancer model, the ⁶⁴Cu-DOTA-ECL1i tracerdemonstrated sensitive and specific detection of tumor cells.Experiments are being performed to correlate the PET imaging data to theprogression of tumors.

As such, the applicability of ⁶⁴Cu-DOTA-ECL1i in detecting CCR2 has beendemonstrated herein in multiple animal models of CCR2 associateddiseases, disorders, and conditions. CCR2 is an interesting target fornumerous diseases (e.g., atherosclerosis, metabolic disease (type IIdiabetes), cancer, multiple sclerosis, rheumatoid arthritis, pain,pulmonary fibrosis). The disclosed imaging agents can be useful for theevaluation of the treatment for CCR2 associated diseases, disorders, orconditions to optimize the treatment strategy and to improve thetherapeutic efficacy.

The imaging agent, as described herein, can be a biocompatible imagingagent. As such, the imaging agent can be stored in or prepared in abuffer of a physiologically relevant pH. For example, the pH can beabout 1 to 3 (e.g., for stomach); about 4 to 7 (e.g., for smallintestine); about 7-8.5 (e.g., for large intestine); about 7.4 (e.g.,for blood pH); about 7.35 (e.g., for CSF); or about 5 to 6 (e.g., forurine pH). As another example, the pH can be about 1; about 1.5; about2; about 2.5; about 3; about 3.5; about 4; about 4.5; about 5; about5.5; about 6; about 6.5; about 6.6; about 6.7; about 6.8; about 6.9;about 7; about 7.1; about 7.2; about 7.3; about 7.4; about 7.5; about 8;or about 8.5.

CCR2 Binding Peptide

As described herein, the imaging agent comprises a CCR2 binding peptide.The CCR2 binding peptide can be any peptide with CCR2 activity. Forexample, the CCR2 binding peptide can be a CCR2 non-competitiveantagonist peptide. As another example, the CCR2 binding peptide can bea cyclized CCR2 binding peptide.

As described herein, monocyte chemo-attractant protein chemokines aresecreted by a wide variety of cell types under a range of inflammatoryconditions such as atherosclerosis, neurodegenerative disease, andvarious forms of cancer (among various other diseases and conditions).One of the most prominent chemokines is monocyte chemoattractantprotein-1 (MCP-1), now called CCL2, which significantly regulatesmigration and infiltration of monocytes to the site of inflammation,predominantly through CC-chemokine receptor 2 (CCR2).

CCR2 binding peptides can be any CCR2 peptide known, such as thosedescribed in US Pat Pub No. 2015/0011477, incorporated herein byreference in its entirety. The imaging agent as described hereincomprises a CCR2 binding peptide. For example, the CCR2 binding peptidecan comprise an ECL1i peptide, as described in US Pat Pub No.2015/0011477, incorporated herein by reference. Because CCR2⁺ cellsmigrate in response to CCL2, CCR2 can be a surrogate marker for CCR2. Assuch, CCL2 and CCR2 can be used interchangeably.

For example, the binding peptide can comprise ECL1i. An ECL1i peptidecan be of the sequence DLeu-Gly-DThr-DPhe-DLeu-DLys-DCys (SEQ ID NO: 3).As another example, a CCR2 binding peptide can be a peptide comprisingthe following amino acid sequence Thr-Phe-Leu-Lys orThr-Phe-Leu-Lys-Cys, useful as a CCR2 non-competitive antagonistpeptide. As another example, the ECL1i peptide can be a cyclized ECL1ipeptide (e.g., Cyclo-(Orn-LGTFLK)).

Among all the peptides tested, the heptapeptide LGTFLKC, named ECL1 (C)inverso, presented interesting properties as a CCR2 non-competitiveantagonist peptide. This peptide corresponds to an inverted sequence inthe third transmembrane domain of CCR2, more precisely in thejuxtamembranous and N-terminal region of the third transmembrane domain.In some embodiments, all or part of the amino acids are in a Dconfiguration.

As another example, as described in US Pat Pub No. 2015/0011477(incorporated by reference, herein), the CCR2 binding peptide can be:

TABLE 5 CCR2 binding peptides SEQ ID NO: Description Peptide SequenceComments 1 Thr Phe Leu Lys Cys 2 Xaa Thr Phe Leu LysXaa in position 1 is absent, is Cys Xaa glycine or represents anamino acid sequence selected from the group consisting ofLG, YLG, and HYLG, “Xaa in position 7” independently isabsent, is methionine, or represents an amino acidsequence selected from the group consisting of MA, MAN,MANG, MANGF, MANGFV, MANGFVW, MANGFVWE, and MANGFVWEN 3 ECL1(C)Leu Gly Thr Phe Leu inverso, Lys Cys ECL1i 4 His Tyr Leu Gly ThrPhe Leu Lys Cys Met Ala 5 Leu Gly Thr Phe Leu Lys Cys Met Ala 6His Tyr Leu Gly Thr Phe Leu Lys Cys 7 Gly Thr Phe Leu LysCys Met Ala Asn Gly Phe 8 Thr Phe Leu Lys Cys Met Ala Asn Gly Phe Val 9His Tyr Leu Gly Thr Phe Leu Lys Cys Met Ala Asn Gly Phe Val Trp 10ECL1 (C) Cys Lys Leu Phe Thr Gly Leu 11 ECL2 (N) Leu Phe Thr Lys Cys 12ECL2 (N) Cys Lys Thr Phe Leu inverso 13 ECL3 (C) His Thr Leu Met ArgAsn Leu 14 ECL3 (C) Leu Asn Arg Met Leu inverso Thr His 15 ECL3 (N)Leu Asn Thr Phe Gln Glu Phe 16 ECL3 (N) Phe Glu Gln Phe Thr inversoAsn Leu 17 Thr Phe Leu Lys 18 Xaa Thr Phe Leu LysXaa in position 1 is absent, is Xaa glycine or represents anamino acid sequence selected from the group consisting ofAG, LG, YLG and HYLG, “Xaa in position 6” independently isabsent or is alanine 19 Leu Gly Thr Phe Leu Lys 20 Ala Gly Thr Phe LeuLys Cys 21 Leu Gly Thr Phe Leu Lys Ala 22 Gly Thr Phe Leu Lys 23Ala Gly Thr Phe Leu Lys Ala 24 Met Ala Asn Gly 25 Met Ala Asn Gly Phe 26Met Ala Asn Gly Phe Val 27 Met Ala Asn Gly Phe Val Trp 28Met Ala Asn Gly Phe Val Trp Glu 29 Met Ala Asn Gly Phe Val Trp Glu Asn30 His Tyr Leu Gly

The CCR2 binding peptide as described herein can comprise an amino acidlength of about 4 amino acids to about 200 amino acids or about 4 aminoacids to about 50 amino acids. For example, the CCR2 biding peptide cancomprise an amino acid length of no more than 4 amino acids; 5 aminoacids; 6 amino acids; 7 amino acids; 8 amino acids; 9 amino acids; 10amino acids; 11 amino acids; 12 amino acids; 13 amino acids; 14 aminoacids; 15 amino acids; 16 amino acids; 17 amino acids; 18 amino acids;19 amino acids; 20 amino acids; 21 amino acids; 22 amino acids; 23 aminoacids; 24 amino acids; 25 amino acids; 26 amino acids; 27 amino acids;28 amino acids; 29 amino acids; 30 amino acids; 31 amino acids; 32 aminoacids; 33 amino acids; 34 amino acids; 35 amino acids; 36 amino acids;37 amino acids; 38 amino acids; 39 amino acids; 40 amino acids; 41 aminoacids; 42 amino acids; 43 amino acids; 44 amino acids; 45 amino acids;46 amino acids; 47 amino acids; 48 amino acids; 49 amino acids; 50 aminoacids; 51 amino acids; 52 amino acids; 53 amino acids; 54 amino acids;55 amino acids; 56 amino acids; 57 amino acids; 58 amino acids; 59 aminoacids; 60 amino acids; 61 amino acids; 62 amino acids; 63 amino acids;64 amino acids; 65 amino acids; 66 amino acids; 67 amino acids; 68 aminoacids; 69 amino acids; 70 amino acids; 71 amino acids; 72 amino acids;73 amino acids; 74 amino acids; 75 amino acids; 76 amino acids; 77 aminoacids; 78 amino acids; 79 amino acids; 80 amino acids; 81 amino acids;82 amino acids; 83 amino acids; 84 amino acids; 85 amino acids; 86 aminoacids; 87 amino acids; 88 amino acids; 89 amino acids; 90 amino acids;91 amino acids; 92 amino acids; 93 amino acids; 94 amino acids; 95 aminoacids; 96 amino acids; 97 amino acids; 98 amino acids; 99 amino acids;100 amino acids; 101 amino acids; 102 amino acids; 103 amino acids; 104amino acids; 105 amino acids; 106 amino acids; 107 amino acids; 108amino acids; 109 amino acids; 110 amino acids; 111 amino acids; 112amino acids; 113 amino acids; 114 amino acids; 115 amino acids; 116amino acids; 117 amino acids; 118 amino acids; 119 amino acids; 120amino acids; 121 amino acids; 122 amino acids; 123 amino acids; 124amino acids; 125 amino acids; 126 amino acids; 127 amino acids; 128amino acids; 129 amino acids; 130 amino acids; 131 amino acids; 132amino acids; 133 amino acids; 134 amino acids; 135 amino acids; 136amino acids; 137 amino acids; 138 amino acids; 139 amino acids; 140amino acids; 141 amino acids; 142 amino acids; 143 amino acids; 144amino acids; 145 amino acids; 146 amino acids; 147 amino acids; 148amino acids; 149 amino acids; 150 amino acids; 151 amino acids; 152amino acids; 153 amino acids; 154 amino acids; 155 amino acids; 156amino acids; 157 amino acids; 158 amino acids; 159 amino acids; 160amino acids; 161 amino acids; 162 amino acids; 163 amino acids; 164amino acids; 165 amino acids; 166 amino acids; 167 amino acids; 168amino acids; 169 amino acids; 170 amino acids; 171 amino acids; 172amino acids; 173 amino acids; 174 amino acids; 175 amino acids; 176amino acids; 177 amino acids; 178 amino acids; 179 amino acids; 180amino acids; 181 amino acids; 182 amino acids; 183 amino acids; 184amino acids; 185 amino acids; 186 amino acids; 187 amino acids; 188amino acids; 189 amino acids; 190 amino acids; 191 amino acids; 192amino acids; 193 amino acids; 194 amino acids; 195 amino acids; 196amino acids; 197 amino acids; 198 amino acids; 199 amino acids; or 200amino acids. Recitation of each of these discrete values is understoodto include ranges between each value. Recitation of each of a range isunderstood to include discrete values within the range.

Radiolabel

The imaging agent, as described herein, comprises a radiolabel (alsoknown as a radionuclide). Radiolabeling processes are well known andalso described in Example 1; see e.g. Fani et al. Theranostics 2012;2(5):481-501. doi:10.7150/thno.4024. Except as otherwise noted herein,therefore, the process of the present disclosure can be carried out inaccordance with such processes.

One embodiment of the present disclosure provides for a radiolabeledpeptide. According to another embodiment, the radiolabeled compound canbe an imaging agent.

References herein to “radiolabeled” include a compound where one or moreatoms are replaced or substituted by an atom having an atomic mass ormass number different from the atomic mass or mass number typicallyfound in nature (i.e., naturally occurring). One non-limiting exceptionis ¹⁹F, which allows detection of a molecule which contains this elementwithout enrichment to a higher degree than what is naturally occurring.Compounds carrying the substituent ¹⁹F may thus also be referred to as“labelled” or the like. The term radiolabeled may be interchangeablyused with “isotopically-labelled”, “labelled”, “isotopic tracer group”,“isotopic marker”, “isotopic label”, “detectable isotope”, or“radioligand”.

In one embodiment, the compound comprises one or more radiolabeledgroups.

Examples of suitable, non-limiting radiolabel groups can include: ²H (Dor deuterium), ³H (T or tritium), ¹¹C, ¹³C, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ¹³N,¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ⁸⁹Sr, ³⁵S, ¹⁵³Sm, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Tl^(99m)Tc, ⁹⁰Y, or ⁸⁹Zr. It is to be understood that an isotopicallylabeled compound needs only to be enriched with a detectable isotope to,or above, the degree which allows detection with a technique suitablefor the particular application, e.g., in a detectable compound labeledwith ¹¹C, the carbon-atom of the labeled group of the labeled compoundmay be constituted by ¹²C or other carbon-isotopes in a fraction of themolecules. The radionuclide that is incorporated in the radiolabeledcompounds will depend on the specific application of that radiolabeledcompound. For example, “heavy” isotope-labeled compounds (e.g.,compounds containing deuterons/heavy hydrogen, heavy nitrogen, heavyoxygen, heavy carbon) can be useful for mass spectrometric and NMR basedstudies. As another example, for in vitro labelling or in competitionassays, compounds that incorporate ³H, ¹⁴C, or ¹²⁵I can be useful. Forin vivo imaging applications ¹¹C, ¹³C, ¹⁸F, ¹⁹F, ¹²⁰I, ¹²³I, ¹³¹I, ⁷⁵Br,or ⁷⁶Br can generally be useful. In one embodiment, the radiolabel is⁶⁴Cu.

As another example, the imaging agent comprising a radiolabel cancomprise Oxygen-15 water, Nitrogen-13 ammonia, [⁸²Rb] Rubidium-82chloride, [¹¹C], [¹¹C] 25B-NBOMe, [¹⁸F] Altanserin, [¹¹C] Carfentanil,[¹¹C] DASB, [¹¹C] DTBZ, [¹⁸F]Fluoropropyl-DTBZ, [¹¹C] ME@HAPTHI, [¹⁸F]Fallypride, [¹⁸F] Florbetaben, [¹⁸F] Flubatine, [¹⁸F] Fluspidine, [¹⁸F]Florbetapir, [¹⁸F] or [¹¹C] Flumazenil, [¹⁸F] Flutemetamol, [¹⁸F]Fluorodopa, [¹⁸F] Desmethoxyfallypride, [¹⁸F] Mefway, [¹⁸F] MPPF, [¹⁸F]Nifene, Pittsburgh compound B, [¹¹C] Raclopride, [¹⁸F] Setoperone, [¹⁸F]or [¹¹C] N-Methylspiperone, [¹¹C] Verapamil, [¹¹C] Martinostat,Fludeoxyglucose (¹⁸F)(FDG)-glucose analogue, [¹¹C] Acetate, [¹¹C]Methionine, [¹¹C] Choline, [¹⁸F] Fluciclovine, [¹⁸F] Fluorocholine,[¹⁸F] FET, [¹⁸F] FMISO, [¹⁸F] 3′-fluoro-3′-deoxythymidine, [⁶⁸Ga]DOTA-pseudopeptides, [⁶⁸Ga] PSMA, or [¹⁸F] Fluorodeoxysorbitol (FDS).

Chelator

As described herein, radionuclides can be chelated by any method knownin the art.

Processes of chelating a radioligand are well known; see e.g. Andersonet al., Cancer Biother Radiopharm. 2009 August; 24(4): 379-393;Stockholf et al., Pharmaceuticals (Basel). 2014 April; 7(4): 392-418.Except as otherwise noted herein, therefore, the process of the presentdisclosure can be carried out in accordance with such processes. Forexample, chelators for a radiolabel (e.g., ⁶⁴Cu) can be any of thoseknown in the art (e.g., a macrocyclic chelator). As another example, thechelator can comprise NHS-MAG₃, MAG₃, DTPA, 3p-C-NE3TA, 3p-C-NOTA,3p-C-DE4TA, ATSM, tetraazamacrocyclic ligands (e.g., DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTA-NHS,pSCN-Bn-DOTA, pNH₂-Bn-DOTA, TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid,TETA-octreotide (OC)), hexaazamacrobicyclic cage-type ligands (e.g.,Sarcophogine chelators), cross-bridged tetraamine ligands (e.g., CB-TE2A(4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane)),6-Hydrazinopridine-3-carboxylic acid (Hynic), or NHS-Hynic. As anotherexample, a radiolabelled (e.g., ⁶⁴Cu) chelator can be2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA).

Nanoparticle

As described herein, a radiolabel can be doped in or on a nanoparticle,or a radiolabel can be conjugated to a nanoparticle.

The imaging agent, as described herein can comprise any nanoparticleknown in the art suitable for use as an imaging agent. Nanoparticles foruse in molecular probes and imaging agents are well known; see e.g.,Chen et al., Molecular Imaging Probes for Cancer Research, 2012.

Labeling of nanoparticles are well known; see e.g., Yongjian Liu,Michael J Welch, Nanoparticles labeled with positron emission nuclides:advantages, methods, and applications, Bioconjugate Chemistry, 2012, 23,671-682; Stockholf et al., Pharmaceuticals (Basel). 2014 April; 7(4):392-418. Except as otherwise noted herein, therefore, the process of thepresent disclosure can be carried out in accordance with such processes.

For example, a nanoparticle can be a nanocluster or any other type ofnanostructures including organic, inorganic, or lipid nanostructures.

As another example, the nanoparticle can comprise Au or Cu. As anotherexample, the nanoparticle can comprise iron oxide, gold, goldnanoclusters (AuNC), gold nanorods (AuNR), copper (Cu), quantum dots,carbon nanotubes, carbon nanohorn, gadolinium (Gd), dendrimers,dendrons, polyelectrolyte complex (PEC) nanoparticles, calcium phosphatenanoparticles, perfluorocarbon nanoparticles (PFCNPs), or lipid-basednanoparticles, such as liposomes and micelles.

Linker

Described herein are linkers used to attach peptides to a portion of animaging agent (e.g., a core, a nanoparticle, a radiolabel, a chelator,another peptide). A linker can be any composition used for conjugation,for example to a nanoparticle or chelator.

A linker group can be any linker group suitable for use in an imagingagent. Linker groups for imaging agents (e.g., molecular probes) arewell known (see e.g., Werengowska-Ciećwierz et al., Advances inCondensed Matter Physics, Vol. 2015 (2015); Chen et al., Curr Top MedChem. 2010; 10(12): 1227-1236). Except as otherwise noted herein,therefore, the processes of the present disclosure can be carried out inaccordance with such processes.

For example, the linker can conjugate a nanoparticle to a CCR2 bidingpeptide. For example, the CCR2 binding peptide can be covalentlyattached to the linker. For example, the linker can comprise apoly(ethylene glycol) (PEG) derivative. As another example, the linkercan comprise PEG, TA-PEG-Maleimide, TA-PEG-OMe, or TA-PEG. As anotherexample, a linker can comprise an isothiocyanate group, a carboxylicacid or carboxylate groups, a dendrimer, a dendron,Fmoc-protected-2,3-diaminopropanoic acid, ascorbic acid, a silanelinker, minopropyltrimethoxysilane (APTMS), or dopamine. Other covalentcoupling methods can use employ the use of 2 thiol groups, 2 primaryamines, a carboxylic acid and primary amine, maleimide and thiol,hydrazide and aldehyde, or a primary amine and aldehyde. For example,the linker can be an amide, a thioether, a disulfide, anacetyl-hydrazone group, a polycyclic group, a click chemistry (CC) group(e.g., cycloadditions, for example, Huisgen catalytic cycloaddition;nucleophilic substitution chemistry, for example, ring opening ofheterocyclic electrophiles; carbonyl chemistry of the “nonaldol” type,for example, formation of ureas, thioureas, and hydrazones; additions tocarbon-carbon multiple bonds, for example, epoxidation anddihydroxylation); or a physical or chemical bond.

Detecting CCR2/CCL2 (MCP-1) Associated Disease, Disorders, or Conditions

As described herein, the present disclosure provides for methods ofdetecting or imaging CCR2 receptors or evaluating or monitoring a CCR2associated disease, disorder, or condition. CCR2 receptors areupregulated in CCR2 associated disease, disorders, or conditions.

CCR2/CCL2

Because of the relationship between CCL2 and CCR2, a CCR2 associateddisease can also be a disease associated with CCL2 (MCP-1).

Chemokines, or chemotactic cytokines, are small heparin-binding proteinsthat constitute a large family of peptides (60-100 amino acids)structurally related to cytokines, whose main function is to regulatecell trafficking, particularly that of immune cells, and thus are ofrelevance to this BRTC application.

Chemokines can be classified into four subfamilies on the basis of thenumber and location of the cysteine residues at the N-terminus of themolecule and are named CXC, CC, CX3C, and C. They initiate theircellular effects via interaction with a specific G protein-coupledreceptor. Monocyte chemo-attractant protein chemokines are secreted by awide variety of cell types under a range of inflammatory conditions suchas atherosclerosis, neurodegenerative disease and various forms ofcancer. One of the most prominent of these is monocyte chemoattractantprotein-1 (MCP-1), now called CCL2, which significantly regulatesmigration and infiltration of monocytes to the site of inflammation,predominantly through CC-chemokine receptor 2 (CCR2). In the case of themonocyte subsets mentioned above CD16−/Ly6Chi pro-inflammatory monocytesexhibit high CCR2 expression, whereas the CD16+/Ly6Clo low-inflammatorymonocytes do not. This interplay and its impact on monocyte traffickingand tissue inflammation really highlight the importance of CCR2 imagingto identify the critical pro-inflammatory monocyte subset as well aspotentially track its migration from hematopoietic sites to sites ofinflammation in both pre-clinical and clinical research.

CCR2 Associated Diseases, Disorders, or Conditions

A CCR2 associated disease can be any disease, disorder, or condition inwhich CCR2 is involved; the disease disorder, or condition is associatedwith CCR2; or a CCR2 mediated syndrome. CCR2 is implicated inatherosclerosis, prostate cancer, lung transplantation, and lung injury,for example. CCR2 associated diseases can be an inflammatory diseases orcancer. For example, a CCR2 associated disease can be an inflammatorydisease, metabolic disease (e.g., type II diabetes), necrosis,atherosclerosis, cancer (e.g., prostate cancer), multiple sclerosis,atheroma, monocytic leukemia, kidney diseases (e.g.,glomerularnephritis), Hamman-Rich syndrome, endometriosis, rheumatoidarthritis, bronchiolitis, asthma, systemic lupus erythematosus,inflammatory bowel diseases (e.g., colitis), alveolitis, restinosis,brain trauma, psoriasis, idiopathic pulmonary fibrosis, transplantarteriosclerosis, vascular permeability and attraction of immune cellsduring metastasis, a number of different neurological disorders,autoimmune disease, obesity, multiple sclerosis, rheumatoid arthritis,pain, or pulmonary fibrosis.

As another example, the CCR2 associated disease, disorder, or conditioncan be an ophthalmic disorder, uveitis, atherosclerosis, abdominalaortic aneurysm, rheumatoid arthritis, psoriasis, psoriatic arthritis,atopic dermatitis, multiple sclerosis, Crohn's Disease, ulcerativecolitis, nephritis, organ allograft rejection, fibroid lung, renalinsufficiency, diabetes and diabetic complications, diabeticnephropathy, diabetic retinopathy, diabetic retinitis, diabeticmicroangiopathy, tuberculosis, chronic obstructive pulmonary disease,sarcoidosis, invasive staphyloccocia, inflammation after cataractsurgery, allergic rhinitis, acute or chronic sinusitis allergicconjunctivitis, chronic urticaria, asthma, allergic asthma, periodontaldiseases, periodonitis, gingivitis, gum disease, diastoliccardiomyopathies, cardiac infarction, myocarditis, chronic heartfailure, angiostenosis, restenosis, reperfusion disorders,glomerulonephritis, solid tumors and cancers, chronic lymphocyticleukemia, chronic myelocytic leukemia, multiple myeloma, malignantmyeloma, Hodgkin's disease, and carcinomas of the bladder, breast,cervix, colon, head and neck, lung, prostate, or stomach.

As another example, the CCR2 mediated syndrome, disorder, or disease canbe age-related macular degeneration or retinal degeneration. As anotherexample, the CCR2 mediated syndrome, disorder, or disease can be acardiovascular disease, especially ischemia of lower members or of theheart, or atherogenesis. As another example, the CCR2 mediated syndrome,disorder, or disease can be pain, in particular peripheral pain, such aspain from the sciatic nerve. As another example, the CCR2 mediatedsyndrome, disorder, or disease can be acute and chronic lungdiseases-acute lung injury, primary graft dysfunction (PGD) (areperfusion injury after transplant), COPD, asthma, pulmonary fibrosis,bronchiolitis obliterans syndrome, and fungal pneumonia.

CCL2 is also associated with the neuroinflammatory processes that takeplace in various diseases of the central nervous system (CNS), which arecharacterized by neuronal degeneration. CCL2 expression in glial cellsis increased in epilepsy, brain ischemia Alzheimer's diseaseexperimental autoimmune encephalomyelitis (EAE), and traumatic braininjury.

As another example, CCR2 has been shown to be associated with idiopathicanterior uveitis; HIV-1; Cd3zeta deficiency; cytomegalovirus retinitis;rhinoscleroma; secondary progressive multiple sclerosis; lipidpneumonia; rheumatoid arthritis; or macular degeneration, age-related,1.

As another example, CCL2 has been shown to be associated with neuraltube defects; vangl1-related neural tube defect; HIV-1; mycobacteriumtuberculosis, susceptibility to mycobacterium tuberculosis, protectionagainst, included; proliferative glomerulonephritis; arthritis;rheumatoid arthritis; herpes simplex virus keratitis; anthracosis;crescentic glomerulonephritis; peritonitis; acute cystitis;arteriosclerosis; xanthogranulomatous pyelonephritis; mast-cellleukemia; psoriasis; trypanosomiasis; retinal vasculitis; diabeticmacular edema; mesangial proliferative glomerulonephritis; Chagasdisease; demyelinating disease; renal fibrosis; cerebral aneurysms;denture stomatitis; Kawasaki disease; verruciform xanthoma of skin;interstitial lung disease; severe acute respiratory syndrome; diabeticangiopathy; Erdheim-Chester disease; pulmonary alveolar proteinosis;uveitis; extrapulmonary tuberculosis; encephalitis; pneumonia;endometriosis; carotid artery disease; pneumoconiosis; retinal veinocclusion; abdominal aortic aneurysm; viral meningitis;glomerulonephritis; idiopathic interstitial pneumonia; nephrosclerosis;acute proliferative glomerulonephritis; viral encephalitis; pulmonarysarcoidosis; post-thrombotic syndrome; vascular disease; alcoholichepatitis; papillary conjunctivitis; hyperhomocysteinemia; scleritis;radiculopathy; pulmonary fibrosis; lipoid nephrosis; pleuraltuberculosis; autoinflammation, lipodystrophy, and dermatosis syndrome;pleurisy; complex regional pain syndrome; pyelonephritis;endocervicitis; leptospirosis; microvascular complications of diabetes1; dengue shock syndrome; peripheral artery disease; chorioamnionitis;silicosis; pelvic inflammatory disease; vitreoretinopathy, neovascularinflammatory disease; purulent labyrinthitis; stachybotrys chartarum;transient cerebral ischemia; neuritis; keratitis; tuberculousmeningitis; nonspecific interstitial pneumonia; limb ischemia; secondaryprogressive multiple sclerosis; retinal vascular occlusion; Israeli ticktyphus; bacteriuria; pulmonary fibrosis, idiopathic; stromal keratitis;bone cancer; sarcoidosis 1; malaria; ureteral disease; coronary arteryaneurysm; lung disease; macular holes; urinary tract obstruction;extrinsic cardiomyopathy; periodontitis; systemic lupus erythematosus;vasculitis; ariboflavinosis; eye disease; meningitis; artery disease;cystitis; central nervous system disease; macular degeneration,age-related, 1; obesity; multiple sclerosis, disease progression,modifier of; diabetes mellitus, noninsulin-dependent; urinary systemdisease; endometrial stromal sarcoma; myocardial infarction;degeneration of macula and posterior pole; overnutrition; respiratorysystem disease; acquired metabolic disease; or bone inflammationdisease.

CCR2 Associated Lung Disease

Lung diseases are often characterized by the nature of immune orinflammatory cells that are found within the tissues and airways.Chemokines guide the migration and function of inflammatory cellsharboring their cognate receptor. In the lung, the chemokine CCL2(monocyte chemoattractant protein-1, MCP-1), is frequently elevated inacute and chronic lung disease. CCL2 is the major ligand for thechemokine receptor CCR2, which is found largely on immune cells andnotably on monocytes, dendritic cells (DCs), and T cells. As such, theimaging agent, as described herein can be used to image CCR2 in the lungto guide diagnosis and therapy.

As described herein, CCL2/CCR2 is elevated in lung disease. TheCCL2/CCR2 axis is demonstrated to be active in acute and chronic lungdiseases. CCR2 function is supported by deletion or antagonism inrelated mouse models. Diseases include those for which specifictherapies are currently limited:

Acute Lung Injury.

Excessive recruitment of CCR2-dependent leukocytes impacts thepathogenesis of acute lung injury in human ARDS and mouse models,shaping the magnitude and duration of disease. Endotoxin (LPS) triggersCCR2-depedent migration of monocytes to the lung when administeredintratracheally and also influences subsequent neutrophil recruitment inlung.

PGD.

Reperfusion injury immediately following lung transplant, known asprimary graft dysfunction (PGD), is marked by elevated CCL2 levels inBAL fluid, while clinical improvement occurs as CCL2 levels fall. It wasobserved that CCR2 is required for mobilization of CD11b⁺Ly6C^(hi)monocytes and accumulation into lung allographs in a mouse lungtransplant model of PGD.

COPD.

Human studies of CCL2/CCR2 in COPD show increased levels of CCL2 in thesputum, BAL fluid and lungs (including ex-smokers) and expression ofCCR2 on leukocytes and epithelia. It was recently reported thatincreased CCR2 was observed on interstitial monocytes from COPD lungtissue.

Asthma.

In human subjects with asthma studied by segmental allergybronchoprovocation, CCL2 and CCR2 were increased in BAL. Blocking orgenetic deletion of CCL2/CCR2 in mouse models of airway allergensensitization prevents monocyte and dendritic cell migration andallergic responses.

Pulmonary Fibrosis and Others.

Human and mouse studies have also implicated the CCL2/CCR2 axis in thepathogenesis of pulmonary fibrosis, bronchiolitis obliterans syndrome,and fungal pneumonia.

Ischemia-Reperfusion Injury.

Ischemia reperfusion injury-mediated primary graft dysfunctionsubstantially hampers short- and long-term outcomes after lungtransplantation. This condition continues to be diagnosed based onoxygen exchange parameters as well as radiological appearance, andtherapeutic strategies are mostly supportive in nature. Identifyingpatients who may benefit from targeted therapy would therefore be highlydesirable.

Recruitment of innate immune cells to lungs shortly after reperfusionplays a key role in mediating tissue injury. It is reported herein thatin addition to their well-recognized role in promoting acute injury,neutrophils can enhance adaptive immune responses after pulmonarytransplantation. Interestingly, it has recently been demonstrated thatmonocytes facilitate the extravasation of neutrophils into reperfusedlungs. It thus seems that for lung graft dysfunction, monocytes play acritical role in orchestrating tissue injury. These observations may bemore generalizable as CCR2⁺ monocytes promote the transendothelialmigration of neutrophils in murine models of arthritis. Mouse monocytesare heterogeneous with CD11b⁺Ly6C^(high)CCR2^(high) monocytes consideredto be an inflammatory subset. Experiments using CCR2-deficient mice havedemonstrated that CCR2 signaling contributes to myocardial, renal andcerebral ischemia reperfusion injury. Attenuation of injury wasassociated with reductions of monocytic and neutrophilic infiltrationinto the affected tissues.

Here, it has been shown that CCR2 expression in murine lung transplantrecipients promotes monocyte infiltration into pulmonary grafts andmediates graft dysfunction. The development of the new positron emissiontomography imaging agents using a CCR2 binding peptide ECLi1 that can beused to monitor inflammatory responses after organ transplantation hasbeen shown herein. Both ⁶⁴Cu-radiolabeled ECL1i peptide radiotracer(⁶⁴Cu-DOTA-ECL1i) and ECL1i-conjugated gold nanoclusters doped with ⁶⁴Cu(⁶⁴CuAuNCs-ECL1i) showed specific detection of CCR2, which isup-regulated during ischemia-reperfusion injury after lungtransplantation. Due to its fast pharmacokinetics ⁶⁴Cu-DOTA-ECL1ifunctioned efficiently for rapid and serial imaging of CCR2. Themultivalent ⁶⁴CuAuNCs-ECL1i with extended pharmacokinetics is favoredfor long-term CCR2 detection and potential targeted theranostics. Thisimaging can be applicable for diagnostic and therapeutic purposes for awide variety of immune-mediated diseases.

Lung Injury.

The imaging sensitivity of ⁶⁴Cu-DOTA-ECL1i was characterized in the lungLPS injury model. Specifically, following the intratrachealadministration of LPS at low, intermediate and high dose levels tocreate different levels of injury and inflammation in the lungs,⁶⁴Cu-DOTA-ECL1i PET imaging was performed at 24 h post injury followingthe same imaging protocol. PET images showed strong signals in all theinflammatory lungs. The quantitative uptake analysis of the lungscorrelated with the LPS dose levels, which demonstrated the sensitivityof this PET tracer detecting inflammation.

Atherosclerosis.

Atherosclerosis is a chronic, inflammatory disease, which is theunderlying basis for cardiovascular disease. In the mouse apoE^(−/−)atherosclerosis model, ⁶⁴Cu-DOTA-ECL1i PET image clearly showed thespecific uptake at aortic arch where the atherosclerotic plaque waslocated. Longitudinal studies using this PET tracer to track theprogression of plaque have been performed.

CCR2 Antagonists

The CCR2 antagonists, as described herein can be any CCR2 antagonistknown in the art (see e.g., Struthers M, Pasternak A. CCR2 antagonists.Curr Top Med Chem. 2010; 10(13):1278-98).

Several CCR2 antagonists (see e.g., Example 2 and 3) including smallmolecules and antibodies have been used as inhibitors in variousinflammatory diseases, both experimentally and in clinical trials.However, clinical trials have yielded equivocal or negative results.This may be due to recruitment of anti-inflammatory monocyte populationsor other chemokine receptors contributing to inflammation. Moreover,inadequate dosing or duration of therapy due to concerns for toxicity orimpedance of the essential role of CCR2 for immune surveillance maycloud the benefit of antagonists. It would therefore be desirable todevise a non-invasive CCR2 imaging technique, which is not only able tomonitor the degree of receptor occupancy to aid in dose selection, butalso to determine the therapeutic response in real time.

For example, a CCR2 antagonist can be NCT01215279; BMS-741672; BMS22;RS504393 (Tocris); BMS CCR2 28; CCX140-B; sc-202525; ucb-102405;benzimidazoles; SB-380732; AZD-6942; 3-aminopyrrolidines; INCB-003284;PF-6309; PF-04136309; PF-04634817; cenicriviroc; AZD2423; or CCR2antibodies. As another example, the CCR2 antagonists can be anantagonist selected from the compounds below.

Company Compound Roche/Iconix

Millennium/ Pfizer

SmithKline

AstraZeneca AZD-6942 Merck

Teijin/BMS

Telik

Incyte

Compound Structure Takeda

Chemokine Therapeutics

Pfizer

Ono

Ono

Merck

Warner-Lambert

Chemokines (e.g., MCP-1 CCL2)

The present disclosure provides the first imaging probe available forCCR2 detection. As described herein, the CCR2 imaging specificity andsensitivity have been well characterized in pre-clinical studies.

Chemokines, or chemotactic cytokines, are small heparin-binding proteinsthat constitute a large family of peptides (60-100 amino acids)structurally related to cytokines, whose main function is to regulatecell trafficking, particularly that of immune cells, and thus are ofrelevance to this BRTC application. Chemokines can be classified intofour subfamilies on the basis of the number and location of the cysteineresidues at the N-terminus of the molecule and are named CXC, CC, CX3C,and C. They initiate their cellular effects via interaction with aspecific G protein-coupled receptor. Monocyte chemo-attractant proteinchemokines are secreted by a wide variety of cell types under a range ofinflammatory conditions such as atherosclerosis, neurodegenerativedisease and various forms of cancer. One of the most prominent of theseis monocyte chemoattractant protein-1 (MCP-1), now called CCL2, whichsignificantly regulates migration and infiltration of monocytes to thesite of inflammation, predominantly through CC-chemokine receptor 2(CCR2). In the case of the monocyte subsets mentioned aboveCD16−/Ly6C^(hi) pro-inflammatory monocytes exhibit high CCR2 expression,whereas the CD16+/Ly6C^(lo) low-inflammatory monocytes do not. Thisinterplay and its impact on monocyte trafficking and tissue inflammationreally highlight the importance of CCR2 imaging to identify the criticalpro-inflammatory monocyte subset as well as potentially track itsmigration from hematopoietic sites to sites of inflammation in bothpre-clinical and clinical research.

CCR2 directs monocytes and other immune cell recruitment in the lung. Amajor role for the CCL2/CCR2 pair is the recruitment of inflammatorymonocytes from the bone marrow and regulation of macrophage, dendriticand T cells maturation. In response to CCL2, CCR2⁺ monocytes adhere tothe vascular endothelial surface and migrate into tissue, alongchemotactic gradients. Inflammatory monocytes (mouse Ly6C^(hi)Ly6G^(lo), human CD14⁺CD16⁻) serve as precursors for classicalmacrophages and conventional DCs. CCR2⁺ monocytes also provide asecondary source of proinflammatory modulators, such as tumor necrosisfactor-α, interleukin-1β and matrix metalloproteinases, contributing tolung injury. Although inflammatory monocytes are essential earlyresponders, excessive or prolonged recruitment impairs resolution ofinflammation and propagates disease progression.

Molecular Engineering

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

Nucleotide and/or amino acid sequence identity percent (%) is understoodas the percentage of nucleotide or amino acid residues that areidentical with nucleotide or amino acid residues in a candidate sequencein comparison to a reference sequence when the two sequences arealigned. To determine percent identity, sequences are aligned and ifnecessary, gaps are introduced to achieve the maximum percent sequenceidentity. Sequence alignment procedures to determine percent identityare well known to those of skill in the art. Often publicly availablecomputer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR)software is used to align sequences. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared. When sequences are aligned, the percentsequence identity of a given sequence A to, with, or against a givensequence B (which can alternatively be phrased as a given sequence Athat has or comprises a certain percent sequence identity to, with, oragainst a given sequence B) can be calculated as: percent sequenceidentity=X/Y100, where X is the number of residues scored as identicalmatches by the sequence alignment program's or algorithm's alignment ofA and B and Y is the total number of residues in B. If the length ofsequence A is not equal to the length of sequence B, the percentsequence identity of A to B will not equal the percent sequence identityof B to A.

Generally, conservative substitutions can be made at any position solong as the required activity is retained. So-called conservativeexchanges can be carried out in which the amino acid which is replacedhas a similar property as the original amino acid, for example theexchange of Glu by Asp, Gin by Asn, Val by lie, Leu by lie, and Ser byThr. For example, amino acids with similar properties can be Aliphaticamino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine);Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine,Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids(e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine,Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); orAcidic and their Amide (e.g., Aspartate, Glutamate, Asparagine,Glutamine). Deletion is the replacement of an amino acid by a directbond. Positions for deletions include the termini of a polypeptide andlinkages between individual protein domains. Insertions areintroductions of amino acids into the polypeptide chain, a direct bondformally being replaced by one or more amino acids. Amino acid sequencecan be modulated with the help of art-known computer simulation programsthat can produce a polypeptide with, for example, improved activity oraltered regulation. On the basis of this artificially generatedpolypeptide sequences, a corresponding nucleic acid molecule coding forsuch a modulated polypeptide can be synthesized in-vitro using thespecific codon-usage of the desired host cell.

Formulation

The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in, for example, Remington'sPharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), incorporated herein by reference in its entirety.Such formulations will contain a therapeutically effective amount of abiologically active agent described herein, which can be in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject.

The term “formulation” refers to preparing a drug in a form suitable foradministration to a subject, such as a human. Thus, a “formulation” caninclude pharmaceutically acceptable excipients, including diluents orcarriers.

The term “pharmaceutically acceptable” as used herein can describesubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. Examplesof pharmaceutically acceptable ingredients can be those havingmonographs in United States Pharmacopeia (USP 29) and National Formulary(NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md.,2005 (“USP/NF”), or a more recent edition, and the components listed inthe continuously updated Inactive Ingredient Search online database ofthe FDA. Other useful components that are not described in the USP/NF,etc. may also be used.

The term “pharmaceutically acceptable excipient,” as used herein, caninclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, or absorption delaying agents. The useof such media and agents for pharmaceutical active substances is wellknown in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofaras any conventional media or agent is incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

A “stable” formulation or composition can refer to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

The formulation should suit the mode of administration. The agents ofuse with the current disclosure can be formulated by known methods foradministration to a subject using several routes which include, but arenot limited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

Agents or compositions described herein can also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic and Monitoring or Evaluation Methods

Also provided is a process of treating, evaluating, or monitoring aCCR2/CCL2 associated disease, disorder, or condition in a subject inneed administration of a therapeutically effective amount of atherapeutic agent (e.g., a CCR2 antagonist), so as to substantiallyinhibit a CCR2/CCL2 associated disease, disorder, or condition, slow theprogress of a CCR2/CCL2 associated disease, disorder, or condition, orlimit the development of a CCR2/CCL2 associated disease, disorder, orcondition.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the evaluation or therapeutic methodsdescribed herein can be a subject having, diagnosed with, suspected ofhaving, or at risk for developing a CCR2/CCL2 associated disease,disorder, or condition. A determination of the need for treatment willtypically be assessed by a history and physical exam consistent with thedisease or condition at issue. Diagnosis of the various conditionstreatable by the methods described herein is within the skill of theart. The subject can be an animal subject, including a mammal, such ashorses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters,guinea pigs, and chickens, and humans. For example, the subject can be ahuman subject.

Generally, a safe and effective amount of a therapeutic agent or animaging agent is, for example, that amount that would cause the desiredtherapeutic or imaging effect in a subject while minimizing undesiredside effects. In various embodiments, an effective amount of atherapeutic agent described herein can substantially inhibit a CCR2/CCL2associated disease, disorder, or condition, slow the progress of aCCR2/CCL2 associated disease, disorder, or condition, or limit thedevelopment of a CCR2/CCL2 associated disease, disorder, or condition.

According to the methods described herein, administration can beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of an imaging or therapeutic agent can be employed inpure form or, where such forms exist, in pharmaceutically acceptablesalt form and with or without a pharmaceutically acceptable excipient.For example, the compounds of the present disclosure can beadministered, at a reasonable benefit/risk ratio applicable to anymedical treatment, in a sufficient amount to evaluate a CCR2/CCL2associated disease, disorder, or condition, substantially inhibit aCCR2/CCL2 associated disease, disorder, or condition, slow the progressof a CCR2/CCL2 associated disease, disorder, or condition, or limit thedevelopment of a CCR2/CCL2 associated disease, disorder, or condition.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level or dose level to beused as an imaging agent for any particular subject will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the subject; the time of administration; the route ofadministration; the rate of excretion of the composition employed; theduration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics:The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^(th) ed.,Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) AppliedBiopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN0071375503). For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved. If desired, the effectivedaily dose may be divided into multiple doses for purposes ofadministration. Consequently, single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present disclosure will be decided by an attendingphysician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, can benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingcan also include inhibiting the state, disease, disorder, or condition,e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Administration of an imaging agent or a therapeutic agent can occur as asingle event or over a time course of treatment. For example, an imagingagent or a therapeutic agent can be administered daily, weekly,bi-weekly, or monthly. For treatment of acute conditions, the timecourse of treatment will usually be at least several days. Certainconditions could extend treatment from several days to several weeks.For example, treatment could extend over one week, two weeks, or threeweeks. For more chronic conditions, treatment could extend from severalweeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performedprior to, concurrent with, or after conventional treatment modalitiesfor a CCR2/CCL2 associated disease, disorder, or condition.

A therapeutic agent can be administered simultaneously or sequentiallywith another agent, such as an antibiotic, an anti-inflammatory, oranother agent. For example, a therapeutic agent can be administeredsimultaneously with another agent, such as an antibiotic or ananti-inflammatory. Simultaneous administration can occur throughadministration of separate compositions, each containing one or more ofa therapeutic agent, an antibiotic, an anti-inflammatory, or anotheragent. Simultaneous administration can occur through administration ofone composition containing two or more of a therapeutic agent, anantibiotic, an anti-inflammatory, or another agent. A therapeutic agentcan be administered sequentially with an antibiotic, ananti-inflammatory, or another agent. For example, a therapeutic agentcan be administered before or after administration of an antibiotic, ananti-inflammatory, or another agent.

Administration

The imaging agents as described herein can be administered in a subjectin an amount effective to produce images in a variety of imagingmodalities, such as PET or SPECT. Administration and calculations ofamounts of imaging agents are well known in the art and also describedin Examples 4 and 5 (see e.g., Long et al., The Chemistry of MolecularImaging, Wiley, 2014; Saini et al., Spect and MRI Imaging Agents: Brainand Tumor Imaging, Lambert, 2016; Smith et al., Diagnostic Imaging forPharmacists, APA, 2012). Except as otherwise noted herein, therefore,the process of the present disclosure can be carried out in accordancewith such processes.

Agents and compositions described herein can be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition can be used either as exogenous materials or asendogenous materials. Exogenous agents are those produced ormanufactured outside of the body and administered to the body.Endogenous agents are those produced or manufactured inside the body bysome type of device (biologic or other) for delivery within or to otherorgans in the body.

Agents and compositions described herein can be administered in avariety of methods well known in the arts. For example, administrationcan be parenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration. As another example,administration can include, for example, methods involving oralingestion, direct injection (e.g., systemic or stereotactic),implantation of cells engineered to secrete the factor of interest,drug-releasing biomaterials, polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,implantable matrix devices, mini-osmotic pumps, implantable pumps,injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm),nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm),reservoir devices, a combination of any of the above, or other suitabledelivery vehicles to provide the desired release profile in varyingproportions. Other methods of controlled-release delivery of agents orcompositions will be known to the skilled artisan and are within thescope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition can beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system can be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to nontarget tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Kits

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited toimaging agents, nanoparticles, peptides (e.g., CCR2 binding peptides),chelators, radiolabelled compositions, buffers. Such packaging of thecomponents separately can, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the composition. The pack may, for example, comprise metal orplastic foil such as a blister pack. Such packaging of the componentsseparately can also, in certain instances, permit long-term storagewithout losing activity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

Examples

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1: Noninvasive Imaging of CCR2⁺ Cells in Ischemia ReperfusionInjury after Lung Transplantation

Here, we show that CCR2 expression in murine lung transplant recipientspromotes monocyte infiltration into pulmonary grafts and mediates graftdysfunction. We have developed new positron emission tomography imagingagents using a CCR2 binding peptide ECLi1 that can be used to monitorinflammatory responses after organ transplantation. Both⁶⁴Cu-radiolabeled ECL1i peptide radiotracer (⁶⁴Cu-DOTA-ECL1i) (see e.g.,FIG. 1) and ECL1i-conjugated gold nanoclusters doped with ⁶⁴Cu(⁶⁴CuAuNCs-ECL1i) (see e.g., FIG. 5A-FIG. 5C) showed specific detectionof CCR2, which is up-regulated during ischemia-reperfusion injury afterlung transplantation. Due to its fast pharmacokinetics ⁶⁴Cu-DOTA-ECL1ifunctioned efficiently for rapid and serial imaging of CCR2. Themultivalent ⁶⁴CuAuNCs-ECL1i with extended pharmacokinetics is favoredfor long-term CCR2 detection and potential targeted theranostics. Thisdata shows that the use of these imaging agents can be applicable fordiagnostic and therapeutic purposes for a wide variety ofimmune-mediated diseases.

As described in more detail below, in the wild type (WT) to wild typemouse lung transplantation model, ⁶⁴Cu-DOTA-ECL1i tracer was injectedvia tail vein at 1 h post transplantation and 0-60 min dynamic PETimaging was conducted right after the injection. In the donor lung of WTrecipient, strong PET signal was detected, which was nearly 3 timeshigher than the weak accumulation observed in the native lung. Toconfirm the targeting specificity, PET imaging was performed in WT mouseto CCR2 knock-out (CCR2^(−/−)) mouse lung transplantation model. It wasfound out that PET signal was only detected in the donor lung from theWT mouse, which confirmed the CCR2 imaging specificity. Moreover, wecarried out the CCR2 receptor competitive blocking study by injecting⁶⁴Cu-DOTA-ECL1i and excess amount of ECL1i peptide (ECL1i vs.⁶⁴Cu-DOTA-ECL1i molar ratio=500:1) in the wild type to wild type mouselung transplantation model. Quantitative PET data analysis showedsignificantly blocked signal at donor lung compared to uptake in thedonor lung without the blocking agent, indicating the CCR2 specifictracer uptake. Besides PET imaging, flow cytometry andimmunohistochemistry both demonstrated the over-expression of CCR2receptor on the donor lungs collected from the WT to WT transplantationmodel, which confirmed the PET imaging results.

Ischemia reperfusion injury-mediated primary graft dysfunction continuesto represent one of the most serious complications after lungtransplantation. It does not only contribute to early morbidity andmortality, but has also been shown to be a risk factor for thedevelopment of chronic allograft dysfunction (2). Primary graftdysfunction is diagnosed and its severity graded based on the impairmentof oxygen exchange and the presence of infiltrates on chest radiographs.Clearly, the identification of reliable biomarkers could facilitate atimely diagnosis. Moreover, elucidating pathways that contribute to thepathogenesis of primary graft dysfunction will allow for the developmentof targeted therapies. We show that recruitment of CCR2⁺ cells promotespulmonary graft dysfunction. Here we have used a new PET probe againstCCR2 to image lung grafts during ischemia reperfusion injury. This studydemonstrates that infiltration of CCR2⁺ cells into lung grafts can bedetected noninvasively and serially using PET imaging.

Clinically, correlations exist between plasma levels of inflammatorycytokines and chemokines and the development of primary graftdysfunction in lung transplant recipients (19-21). MCP-1, a ligand forCCR2, has been found to be elevated in patients, who suffered from thiscomplication. We show that recruitment of CD11b⁺Ly6C^(hi) monocytes topulmonary grafts is significantly reduced when recipients are deficientin CCR2. Based on our previous observation that monocytes mediatetransendothelial migration of neutrophils in injured lungs, we speculatethat amelioration of ischemia reperfusion injury in CCR2-deficientrecipients may in part be due to reduced neutrophilic infiltration (4).Collectively, these clinical and experimental studies indicate that CCR2is an important contributor to ischemia reperfusion injury and thereforecould serve as a biomarker of primary graft dysfunction and also as atherapeutic target.

PET imaging has been used experimentally and clinically by our group andothers to evaluate transplanted organs (15, 18, 22-24). However, to dateno imaging probe has been available to detect the CCR2 receptor in vivo,which is expressed on monocytes and other cell types that are known tomediate inflammatory responses after transplantation (25, 26). Here, weshowed that a CCR2 binding peptide can specifically detect thesereceptors with PET/CT imaging during lung transplant-mediated ischemiareperfusion injury in vivo both as a monovalent peptide tracer and amultivalent nanoplatform (27).

ECL1i has been recently demonstrated to selectively bind CCR2 in anon-competitive way compared to CCL2 ligand in vitro (27). In thisstudy, ⁶⁴Cu-DOTA-ECL1i retention was primarily detected in the lunggraft after transplantation into syngeneic CCR2-deficient recipients,consistent with the presence of donor monocytes in pulmonary grafts.Given the lack of CCR2 receptor in these hosts, we suggest that theminimal localization in native lungs was due to non-specific retention.Thus, if the localization of ⁶⁴Cu-DOTA-ECL1i in the nativeCCR2-deficient lung is due to non-specific background retention, morethan 70% of the observed accumulation in the graft is due toCCR2-mediated uptake, given that ¹⁵O—H₂O imaging did not show adifference in blood flow.

After lung transplantation into wild type recipients the PET/CT imagesshowed considerable accumulation of CCR2⁺ cells in the whole body.Consistent with other inflammatory models our work has shown that CCR2is critical to mobilize CD11b⁺Ly6C^(high) monocytes from the bone marrowafter lung transplantation (28, 29). In some settings CCR2 may promotemonocyte recruitment from the peripheral blood into inflamed sites (30).Our imaging indicates that CCR2⁺ cells are rapidly released into theperiphery after engraftment of lungs. Through competitive receptorblocking, approximately 75% of the PET signal in the grafts was blockedafter transplantation into wild type recipients, resulting in comparableretentions in both native and donor lungs. The consistent resultsbetween CCR2 receptor-specific uptake from the calculation aftertransplantation into CCR2-deficient hosts and the blocking percentageafter engraftment into wild type recipients support the specificity of⁶⁴Cu-DOTA-ECL1i binding CCR2.

In contrast to monovalent ⁶⁴Cu-DOTA-ECL1i peptide tracers, we observedsix-fold higher retention in the blood 1 hour after injection oftargeted ⁶⁴CuAuNCs-ECL1i nanoclusters, confirming the advantage ofmultivalent nanoclusters for improved CCR2 detection (17). Moreimportantly, the targeted ⁶⁴CuAuNCs-ECL1i probe demonstrated specificretention in donor grafts and minimal localization in native lungs. Ifthe low and non-specific accumulation of ⁶⁴CuAuNCs in native lungs isset as background, more than 80% of the accumulation of ⁶⁴CuAuNCs-ECL1iin pulmonary grafts was due to CCR2-mediated uptake, demonstratingspecific and persistent imaging of CCR2. Furthermore, these specificityvalues were significantly higher than those for the ⁶⁴Cu-DOTA-ECL1ipeptide tracer, underscoring the usefulness of multivalent nanoclustersfor enhanced targeting and longitudinal imaging.

In conclusion, PET imaging of CCR2⁺ cells with targeted molecular probescan serve as a biomarker for primary graft dysfunction after lungtransplantation. Additionally, the targeted nanoclusters provide atheranostic platform for image-guided delivery of specific treatment.These imaging approaches could be useful for a variety of CCR2-mediatedinflammatory conditions, both sterile and infectious.

Recipient CCR2 Expression Promotes Monocyte Recruitment to PulmonaryGrafts and Ischemia Reperfusion Injury after Lung Transplantation.

To assess how recipient expression of CCR2 impacts ischemia reperfusioninjury wild type B6 lungs were transplanted into syngeneic wild type orCCR2-deficient recipients. Lack of CCR2 expression in the host resultedin significant amelioration in ischemia reperfusion injury as evidencedby improvement in oxygen exchange (see e.g., FIG. 3A). We nexttransplanted B6 CD45.1 wild type lungs into congenic B6 CD45.2 wild type(see e.g., FIG. 3B) or B6 CD45.2 CCR2-deficient recipients (see e.g.,FIG. 3C) and evaluated recipient (CD45.2) and donor (CD45.1) monocyteswithin the pulmonary grafts 6 hours later. We noticed a substantialreduction in graft infiltration of recipient monocytes (CD11b⁺Ly6C^(hi))when the hosts lacked CCR2 expression. Notably, a small population ofCCR2-expressing monocytes of donor origin was present in wild typegrafts after transplantation into either wild type or CCR2-deficientrecipients. These findings were corroborated by gene expression analysis(see e.g., FIG. 4).

Biodistribution of ⁶⁴Cu-DOTA-ECL1i and ⁶⁴CuAuNCs-ECL1i.

To assess the pharmacokinetics of ⁶⁴Cu-DOTA-ECL1i, biodistribution wasperformed at 1 hour after transplantation of wild type B6 lungs intowild type recipients and showed fast clearance primarily through thekidneys, with minor accumulation in the liver, spleen and negligibleuptake in other organs (see e.g., FIG. 6A), consistent with otherpeptide tracers (14). After transplantation of wild type B6 lungs intowild type B6 recipients, the uptake in the donor lung was more thantwice the uptake in the native lung.

Compared to the monovalent ⁶⁴Cu-DOTA-ECL1i peptide tracer, multivalent⁶⁴CuAuNCs-ECL1i nanoclusters demonstrated extended blood circulation at1 hour after transplantation with significant renal clearance due to thesmall size (see e.g., FIG. 6). The progressive diminution in liveractivity with stable uptake in the gastrointestinal tract indicatedeffective hepatobiliary excretion (17). In contrast to the decreasedactivity in blood pool organs observed with the peptide, thelocalization of the targeted nanoclusters in spleen and bone marrowremained constant throughout the 24 hour imaging period.

In Vivo PET Imaging of ⁶⁴Cu-DOTA-ECL1i Peptide Tracer.

We next wanted to assess whether differences existed between blood flowto the transplanted graft and the native lung. To this end, PET using¹⁵O-water provides a direct physiologic measurement of circulatoryparameters for regional blood and vascular volume (16). PET/CT images of¹⁵O water at 1 hour after transplantation showed similar signals in bothgraft and native lung (see e.g., FIG. 7). Comparable SUV activities wereobserved in grafts and native lungs after transplantation of wild typeB6 lungs into syngeneic wild type hosts, yielding a graft/native lunguptake ratio of 1.09±0.28. In contrast, ⁶⁴Cu-DOTA-ECL1i imaging in wildtype recipients of wild type lungs showed uptake in the graft withminimal signal retained in the native lung at 1 hour aftertransplantation (see e.g., FIG. 8A, FIG. 8B), resulting in agraft/native lung uptake ratio of 3.71±0.44. These results wereindicative of infiltration of CCR2⁺ cells into lung grafts at these timepoints. A PET signal was predominantly observed in B6 wild type lungsafter transplantation into CCR2-deficient recipients, consistent withdonor CCR2⁺ cells being present in these grafts at this time point. Inthe absence of graft infiltration of recipient CCR2⁺ cells, the uptakein these pulmonary grafts was only 40% of that observed in wild typelungs after transplantation into wild type hosts. The activity of⁶⁴Cu-DOTA-ECL1i retained in both lungs measured from background scanswas less than 10% of the uptake acquired after the re-injection of⁶⁴Cu-DOTA-ECL1i at 4 or 24 hours. Thus, the rapid clearance of thispeptide tracer in lungs indicates that it can be used for serial scansin a short time period allowing for the assessment of dynamic expressionof CCR2. A high intensity of PET signal in the kidney also corroboratedthe renal clearance determined by the biodistribution study. PET imagingperformed on naïve mice showed minimal tracer uptake in the lungs (seee.g., FIG. 9).

The uptake in both grafts and native lungs of wild type recipientsprogressively decreased at 4 and 24 hours likely reflecting temporalchanges of CCR2⁺ cell infiltration or expression of the receptor onthese cells. However, the PET intensities in the pulmonary grafts weresignificantly higher than those in the native lungs at these later timepoints. The graft/native lung uptake ratio reached a peak at 4 hours(4.13±0.32) and at 24 hours decreased to a level (3.73±0.45) similar tothat observed at 1 hour. Unlike the 63% decreased uptake in the donorlung in the wild type B6→wild type B6 combination at 24 hours, we foundno significant difference between the three examined time points (1, 4,and 24 hours) in wild type lung grafts after transplantation intoCCR2-deficient recipients. However, at all time-points, the uptake inthe graft was significantly higher than in the native lung reflectingthe presence of CCR2⁺ cells that are carried over with the transplantedlung (see e.g., FIG. 8A, FIG. 8B). When we co-injected non-radiolabeledECL1i, the uptake of ⁶⁴Cu-DOTA-ECL1i in the pulmonary grafts at 1 hourafter transplantation into wild type hosts was significantly decreasedto a level comparable to that in native lungs (see e.g., FIG. 10)resulting in a significant decrease in the graft/native lung uptakeratio (1.17±0.36), indicating binding specificity of the probe.

We have previously reported that ¹⁸F-FDG PET can be used to detect graftrejection after pulmonary transplantation, which has been linked toglucose uptake by graft-infiltrating T cells (18). ¹⁸F-FDG PET/CT showedsignificantly higher uptake in the donor graft than native lung at 1hour after wild type B6→wild type B6 transplantation (see e.g., FIG.11). However, the FDG imaging yielded a graft/native lung uptake ratioof 2.29±0.037, which was significantly lower than the data acquired with⁶⁴Cu-DOTA-ECL1i.

In Vivo PET/CT Imaging of ⁶⁴CuAuNCs-ECL1i Nanoclusters.

Based on the in vivo pharmacokinetics of the multivalent ⁶⁴CuAuNCs-ECL1inanocluster, PET/CT images were acquired at 1, 4 and 24 hours followingengraftment for both CCR2-targeted ⁶⁴CuAuNCs-ECL1i and non-targeted⁶⁴CuAuNCs in the wild type B6→wild type B6 combination. At 4 hours aftertransplantation, uptake of ⁶⁴CuAuNCs-ECL1i was observed in the graftswith minimal accumulation in the native lungs yielding a graft/nativelung uptake ratio of 6.58±0.47 (see e.g., FIG. 12). For non-targeted⁶⁴CuAuNCs, the signal in the graft was significantly lower while theuptake in the native lung was comparable to the targeted counterpart,resulting in a significantly lower graft/native lung ratio (1.51±0.20).Due to their small size and neutral surface charge (see e.g., FIG. 5),both nanoclusters were cleared through the genitourinary system,confirming the pharmacokinetic data. At 24 hours, the targeted⁶⁴CuAuNCs-ECL1i nanocluster showed relatively stable uptake in bothlungs while the signal was somewhat diminished in the pulmonary graftafter injection of non-targeted ⁶⁴CuAuNCs nanoclusters. Notably, thegraft/native lung ratio of ⁶⁴CuAuNCs-ECL1i gradually increased from7.24±0.38 at 4 hours to 8.44±0.54 at 24 hours. These values weresignificantly higher than those obtained with the non-targetedcounterpart.

Mice and Surgical Procedures.

C57BL/6 (B6), B6 CD45.1 and B6 CCR2-deficient mice were purchased fromThe Jackson Laboratories (Bar Harbor, Me.) and maintained inpathogen-free facilities at Washington University. Orthotopic left lungtransplants were performed following 1 hour of storage inlow-potassium-dextran solution at 4° C. unless otherwise specified (13).Arterial blood gases were measured using an iSTAT Portable ClinicalAnalyzer (iSTAT) (FiO₂ 1.0) after clamping the right pulmonary hilum for5 minutes.

PET Imaging.

At 1 hour after transplantation, 0-60 min dynamic PET/CT scan wasperformed following injection of ⁶⁴Cu-DOTA-ECL1i (100 μCi in 100 μLsaline) with microPET Focus 220 (Siemens, Malvern, Pa.) or Inveon PET/CTsystem (Siemens, Malvern, Pa.). For PET/CT imaging at 4 and 24 hoursafter transplantation, a 30-minute background scan (10 min/frame, 3frames) was performed prior to injecting ⁶⁴Cu-DOTA-ECL1i (100 μCi in 100μL saline). The in vivo retention of ⁶⁴Cu-DOTA-ECL1i in the donor lungfrom the previous injection was quantified and subtracted from theuptake value at 4 or 24 hours. For ⁶⁴CuAuNCs-ECL1i, PET/CT was carriedout 1 hour after transplantation. Instead of a dynamic scan, a staticscan was performed at 1, 4 and 24 hours after injection. The PET imageswere reconstructed with the maximum a posteriori algorithm and analyzedby Inveon Research Workplace. The organ uptake was calculated as percentinjected dose per gram (% ID/g) of tissue in three-dimensional regionsof interest without the correction for partial volume effect (14).Competitive PET blocking studies were performed immediately aftertransplantation with co-injection of non-radiolabeled ECL1i and⁶⁴Cu-DOTA-ECL1i (ECL1i: ⁶⁴Cu-DOTA-ECL1i molar ratio=500:1) followed by a0-60 minute dynamic scan. ¹⁸F-FDG (250 μCi in 100 μL saline) PET/CT wasperformed following the same protocol as ⁶⁴Cu-DOTA-ECL1i (15). Tomeasure blood flow changes in lungs caused by the surgical procedure,¹⁵O-water (˜1 mCi) was injected intravenously into mice used for⁶⁴CuNCs-ECL1i imaging at 1 hour after transplantation, followed by a0-10 minute dynamic scan. The relative blood flow change was evaluatedby standardized uptake values (SUVs) (16).

Statistical Analysis.

Group variation is described as mean±SD. Groups were compared using1-way ANOVA with a Bonferroni post-test. Individual group differenceswere determined with use of a 2-tailed Mann-Whitney test. Thesignificance level in all tests was P<0.05. Prism, version 6.07(GraphPad, La Jolla, Calif.), was used for statistical analyses. Errorbars designate standard deviation of the mean unless indicatedotherwise.

Reagents.

Materials were purchased from Sigma-Aldrich (St. Louis, Mo.) and usedwithout further purification unless otherwise stated. The ⁶⁴Cu(half-life=12.7 h, β⁺=17%, β⁻=40%) was produced at the WashingtonUniversity (15). Functionalized poly(ethylene glycol) (PEG) derivativeswere obtained from Intenzyne Technologies (Tampa, Fla.).Maleimido-mono-amide-DOTA was purchased from Macrocyclics (Dallas,Tex.). ECL1i peptide d(LGTFLKC) was customized by CPC Scientific(Sunnyvale, Calif.). Amicon tubes were purchased from EMD Millipore(Billerica, Mass.). The reverse phase-high performance liquidchromatography system was equipped with a UV/VIS detector (Dionex,Sunnyvale, Calif.), a radioactivity detector (B-FC-3200; BioScan Inc.,Poway, Calif.) and a C-18 column (5 mm, 4.6×220 mm; Perkin Elmer,Waltham, Mass.). Polymeric materials were characterized by ¹H and ¹³Cnuclear magnetic resonance spectroscopy using either a Varian 500 MHz orVarian 600 MHz instrument with the residual solvent signal as aninternal reference. Fast protein liquid chromatography was performed inPBS buffer on an ÄKTA system equipped with TSK Gel Guard SW_(XL) column(40×6.0 mm, 7 μm) and G3000SW_(XL) column (300×7.8 mm, 5 μm) connectedin series and UV/VIS (GE) and radioactivity (BioScan Inc.) detectors.

Synthesis and ⁶⁴Cu Radiolabeling of DOTA-ECL1i.

ECL1i (DLeu-Gly-DThr-DPhe-DLeu-DLys-DCys) (1.562 mg, 0.2 μmol) andmaleimido-mono-amide-DOTA (1.573 mg, 0.2 μmol) (Macrocyclics)conjugation was performed in pH 7.4 phosphate buffer at 4° C. overnight(See e.g., FIG. 1). The crude conjugate was purified by HPLC to reach99% chemical purity and characterized by mass spectrometry, whichconfirmed the presence of one DOTA per peptide (M⁺ calculated 1306.65,found: 1306.69, ABI 4700 MALDI TOF-TOF). DOTA-ECL1i (see e.g., FIG. 1,FIG. 2A-FIG. 2B) (10 μg, 7.66 nmol) was incubated with ⁶⁴Cu (2 mCi) in50 μL of 0.1 M pH 5.5 NH₄OAc buffer at 43° C. for 1 hour, with a yieldof 95.6%±2.8% (n=12). The specificity activity of ⁶⁴Cu-DOTA-ECl1i wasdetermined as 261±7.6 μCi/nmol (see e.g., FIG. 5A-FIG. 5C).

Synthesis of TA-PEG-OMe.

A solution of NH₂—PEG750-OMe (0.13 g, 0.17 mmol) in DCM (0.4 mL) wasadded dropwise to a mixture of thioctic acid (0.034 g, 0.17 mmol), DCC(0.035 g, 0.17 mmol) and 4-dimethylaminopyridine (0.0040 g, 0.033 mmol)in DCM (0.8 mL). After overnight stirring, the mixture was filtered andthen rinsed with ethyl acetate. The combined filtrate was dried and theresidue was dissolved in H₂O. The aqueous solution was extracted withdiethyl ether once and saturated with NaHCO₃. The aqueous solution wasextracted with DCM and the organic phase was dried over anhydrous sodiumsulfate. After evaporation of the solvent, the residue was purified bychromatography on silica gel (DCM:ethanol=10:1, v/v) to obtain the finalproduct as a light yellow solid. ¹H NMR (CDCl₃, 400 MHz) δ (ppm): 6.23(bs, 1H), 3.64 (m), 3.53 (m, 4H), 3.43 (m, 2Hs), 3.37 (s, 3Hs), 3.16 (m,1H), 3.12 (m, 1H), 2.46 (sextet, 1H, J=6.4 Hz), 2.19 (t, 2Hs, J=7.6 Hz),1.90 (m, 1H), 1.70 (m, 4Hs), 1.45 (m, 2Hs). ¹³C NMR (CDCl₃, 100 MHz) δ(ppm): 25.37, 28.93, 34.66, 36.32, 38.45, 39.14, 40.22, 56.42, 59.04,69.93, 70.20, 70.51, 70.55, 71.92, 172.77.

TA-PEG-ECL1i.

PBS (pH=7.4 1 mL) was added to TA-PEG-Maleimide (Mw=884.11, 2.42 mg,0.0031 mmol) in a centrifuge vial. When the TA-PEG-Maleimide wascompletely dissolved, the solution was added to ECL1i peptide (2.74 mg,0.0031 mmol). After the mixture was stirred overnight at 4° C., thesolution was purified by RP-HPLC with a H₂O/MeCN solvent system. Theproduct was recovered by lyophilization. MALDI-MS for C₇₅H₁₂₉N₁₁O₂₄S₃:M⁺ calculated: 1663.84; found: 1663.53 (ABI 4700 MALDI TOF-TOF).

Synthesis of Non-Radioactive Nanoclusters.

In a typical reaction, water (0.488 mL), HAuCl₄ (10 mM, 50 μL), andCuCl₂ (1 mM, 5 μL) were mixed in a glass vial, followed by the dropwiseaddition of TA-PEG-OMe (MW=750 Da, 2.5 mM, 600 μL). Sodium borohydride(40 mM, 175 μL) was added to the mixture and rapidly stirred at roomtemperature for 4 hours. The CuAu nanoclusters (CuAuNCs) were purifiedby centrifugation filtration (Amicon, 10K) and washed with pH 7.4phosphate buffer three times.

Synthesis of ⁶⁴CuAuNCs.

The ⁶⁴Cu incorporated AuNCs (⁶⁴CuAuNCs) were prepared following the sameprocedure as described for non-radioactive CuAu nanoclusters. Instead ofadding CuCl₂, radioactive ⁶⁴CuCl₂ (4.2 mCi) was added. The synthesized⁶⁴CuAuNCs was then purified by centrifugation filtration (Amicon, 10K)and washed with phosphate buffer (pH=7.4) three times. The radiochemicalpurity was determined by instant radio-thin layer chromatography(Radio-TLC).

Synthesis of ⁶⁴CuAuNCs-ECL1i.

Water (0.334 mL), HAuCl₄ (10 mM, 25 μL) were mixed in a glass vial,followed by the dropwise addition of TA-PEG-ECL1i (5 mM, 100 μL) andTA-PEG-OMe (M_(w) of PEG=750 Da, 2.5 mM, 100 μL). After addition of⁶⁴CuCl₂ (1.2 mCi), sodium borohydride (40 mM, 100 μL) was added to themixture with rapid stirring at room temperature and then continued forat least 4 hours. The ⁶⁴CuAuNCs-ECL1i were purified by centrifugationfiltration (Amicon, 10K) and washed with phosphate buffer (pH=7.4) threetimes. The radiochemical purity was determined by instant radio-thinlayer chromatography (Radio-TLC) to reach ≧95% radiochemical purity foranimal studies and the specific activity was 1.07 mCi/nmol.

Characterization of DOTA-ECL1i.

For example, a2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA) chelator was conjugated to a cysteineresidue of the peptide (DOTA-ECL1i) in pH 7.4 phosphate buffer. Theconjugate was purified by high performance liquid chromatography andcharacterized by mass spectrometry (see e.g., FIG. 2). The purifiedDOTA-ECL1i was radiolabeled with ⁶⁴Cu (t_(1/2)=12.7 h, β⁺=0.653 Mev(17.8%), β⁻=0.579 Mev (38.4%)) (⁶⁴Cu-DOTA-ECL1i) for PET imaging.

Here, it has been shown that a chelator (e.g.,2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA)) was conjugated to cysteine residue ofa CCR2 binding peptide (DOTA-ECL1i) in pH 7.4 phosphate buffer (seee.g., FIG. 1). The conjugate was purified by high performance liquidchromatography and characterized by mass spectrometry. The purifiedDOTA-ECL1i was radiolabeled with ⁶⁴Cu (t1/2=12.7 h, β+=0.653 Mev(17.8%), β−=0.579 Mev (38.4%)) (⁶⁴Cu-DOTA-ECL1i) for PET imaging. It iscontemplated that the peptide can be conjugated with any radiolabeland/or nanoparticle used for other imaging modalities (e.g., ¹³C, ²H,iron oxide for MRI).

Characterization of CuAuNCs and CuAuNCs-ECL1i.

The UV-Vis absorption spectra were recorded using a Cary 60 UV-Visspectrometer (Agilent Technologies, Santa Clara, Calif.). The ⁶⁴CuAuNCsand ⁶⁴CuAuNCs-ECL1i were examined after radioactive decay using a TecnaiG2 F20 ST Transmission Electron Microscope (TEM) operated at 200 kV(FEI, Hillsboro, Oreg.). The TEM image showed that decayed CuAuNCs-ECL1ihad a uniform size of 2.2±0.6 nm. The hydrodynamic size determined bydynamic light scattering (NanoZS, Malvern, Worcestershire, UK) showed anarrow size distribution of 5.0±0.5 nm with the zeta potential as−6.7±1.5 mV.

Flow Cytometry.

Lung tissue was cut into small pieces and digested by placement into aRPMI 1640 solution containing Type 2 collagenase (1 mg/mL) (WorthingtonBiochemical Corporation, Lakewood, N.J.) and 10 U/mL DNase (Sigma, St.Louis, Mo.) at 37° C. for 60 min. The digested tissue was then passedthrough a 70-μm cell strainer and treated with ACK lysing buffer. Cellswere stained with fluorochrome-labeled anti-CD45.2 (clone 104,eBioscience, San Diego, Calif.), anti-CD45.1 (clone A20, BD Biosciences,San Jose, Calif.), anti-CD11b (clone M1/70, BioLegend, San Diego,Calif.), anti-Ly6C (clone AL-21, BD Biosciences), anti-CCR2 (clone575301, R&D Systems, Minneapolis, Minn.) and isotype control antibodies.

Biodistribution Studies.

C57BL/6 mice were used for the biodistribution studies. About 10 μCi of⁶⁴Cu-DOTA-ECL1i in 100 μL saline (APP pharmaceuticals, Schaumburg, Ill.)were injected via the tail vein. The mice were anesthetized with inhaledisoflurane and re-anesthetized before euthanasia by cervical dislocationat each time point (1 hour after injection, n=4/group). Organs ofinterest were collected, weighed and counted in a Beckman 8000 gammacounter (Beckman, Fullterton, Calif.). Standards were prepared andmeasured along with the samples to calculate the percentage of theinjected dose per gram of tissue (% ID/gram).

Real-Time PCR Assay.

RNA isolated from transplanted mouse lung specimens (right-host lung,left-transplanted graft) was used for real-time RT-PCR. Tissue RNA wasisolated using Nucleospin RNA kits (Macherey-Nagel; Bethlehem, Pa.) perthe manufacturer's instruction. Reverse transcription reactions used 1μg of total RNA, random hexamer priming, and Superscript II reversetranscriptase (Invitrogen). Expression of CCR2 and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were determined using Taqman assays(Invitrogen) and an Eco™ Real-Time PCR System (Illumina, San Diego,Calif.) in duplicate in 48-well plates. PCR cycling conditions were asfollows: 50° C. for 2 min, 95° C. for 21 s, and 60° C. for 20 s. GAPDHexpression was used as a comparator using ΔΔ Ct calculations.

REFERENCES FOR EXAMPLE 1

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Example 2: PET-Based Imaging of Chemokine Receptor-2 in Experimental andDisease-Related Lung Inflammation

The following example describes the characterization of a chemokinereceptor-2 (CCR2) binding peptide adapted for use as a positron emissiontomography (PET) radiotracer for non-invasive detection of lunginflammation in a mouse model of lung injury and in human tissues fromsubjects with lung disease.

In particular, PET images obtained in mouse lungs following injury withLPS, was significantly greater than the saline control group (mean %ID/g=4.43 vs. 0.99; P<0.001). PET signal was significantly diminishedwith blocking studies using non-radiolabeled ECL1i in excess (mean %ID/g=0.63; P<0.001) and in CCR2-deficient mice (mean % ID/g=0.39;P<0.001). The ECL1i signal was associated with an elevated level ofmouse lung monocytes. COPD lung tissue displayed significantly elevatedCCR2 levels compared to non-diseased tissue (median 12.8 vs. 1.2 percentcells/sample; P=0.002), which was detected by ⁶⁴Cu-DOTA-ECL1i usingautoradiography. In summary, ⁶⁴Cu-DOTA-ECL1i performed as a promisingtool for PET-based detection of CCR2-directed inflammation in an animalmodel and in human tissues as a step toward clinical translation.

Lung inflammation is a result of the recruitment of inflammatory cellsalong chemokine gradients, guided by their cognate receptors (1,2). Insome lung conditions, patterns of inflammatory cell recruitment can beused to diagnose diseases and direct therapeutic decisions (e.g.,asthma, eosinophilic pneumonia) (3,4). However, developing a detectionstrategy for identifying immune population signatures has been difficultsince many inflammatory cells are localized within the lung parenchyma,out of reach of conventional diagnostic tools. Consequently,understanding the contribution of immune cell subsets in disease islimited and the clinical development of specific antagonists for lungdisease is stalled. Non-invasive detection could ultimately be used tocharacterize an individual's molecular status, disease activity andresponse to established or new therapies (5).

Chemokine (C-C Motif) ligand 2 (CCL2; also called Monocytechemoattractant protein-1, MCP-1) and its receptor chemokine (C-C motif)receptor 2 (CCR2) are often elevated in lung tissue of subjects withpulmonary diseases (6-10). CCR2 is a surface receptor found on mostinflammatory monocytes and macrophages, as well as some dendritic cellsand lymphocytes (1,2). CCR2⁺ inflammatory monocytes are essential earlyresponding immune cells, and excessive or prolonged recruitment impairsresolution of inflammation and propagates disease progression (3, 11,12). Experimentally, endotoxin triggers CCR2-dependent migration ofmonocytes and macrophages to the lung and influences subsequentneutrophil recruitment (13-15). CCR2⁺ signaling in monocytes provides asecondary source of proinflammatory cytokines and proteases,contributing to lung injury (12,16). Recruitment of CCR2-dependentleukocytes impacts the magnitude and duration of acute respiratorydistress syndrome (17) and elevated numbers of CCR2⁺ monocytes areassociated with ongoing inflammation in chronic obstructive pulmonarydisease (COPD) (6,7), supporting the use of CCR2⁺ cells as a marker ofdisease activity. CCR2⁺ cells may also serve as a therapeutic targetsince CCR2 blockade improves outcome in animal models of disease and hassteered considerable effort toward the development and testing CCR2antagonists (18,19).

We have recently described the use of a peptide that binds the firstextracellular loop (ECL1i) of CCR2 (20) for non-invasive imaging forinitial studies in a lung ischemia-reperfusion model (21), but thegeneralizability to common inflammatory conditions and the activity inhuman disease tissue is unknown. The goal of this study is to furthercharacterize the ECL1i-based PET radiotracer for the non-invasivedetection of monocyte-related inflammation in a mouse model ofendotoxin-induced lung injury and in human lung tissue.

Materials and Methods.

The study had institutional Animal and Human Studies Committees approvaland patient consent. A 7-amino acid CCR2 binding peptide (ECL1i) wasconjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) and labeled with copper-64 (⁶⁴Cu) or fluorescent dye. Lunginflammation was induced by intratracheal administration oflipopolysaccharide (LPS) in wild-type (n=19) and CCR2-deficient mice(n=4), and compared to wild-type mice given control saline (n=5) by PETimaging performed following intravenous injection of ⁶⁴Cu-DOTA-ECL1i.Lung immune cells and those binding fluorescently labeled ECL1i in vivowere detected by flow cytometry. Lung inflammation in tissue fromsubjects with non-diseased lungs donated for lung transplantation (n=1)and chronic obstructive pulmonary disease (COPD) who were undergoinglung transplant (N=16) was evaluated for CCR2 by immunostaining andautoradiography (n=6, COPD) with ⁶⁴Cu-DOTA-ECL1i. Groups were comparedusing ANOVA, Mann Whitney U- or t-tests.

Human Tissues.

The Institutional Review Board approved these studies. Human COPD lungtissue samples were obtained at the time of lung transplantation fromindividuals who provided consent prior to surgery (n=16). All patientsundergoing lung transplantation had very severe COPD by clinicalcriteria. Non-diseased, donated lungs were those not usable fortransplantation or tissues provided after lungs were downsized (n=11).Samples were prospectively collected between 2005 and 2013,de-identified and stored prior to use (TABLE 1). Samples were selectedfor analysis based on tissue availability without pre-selection forspecific clinical features.

TABLE 1 Donor and COPD subjects providing lung tissue for CCR2immunostaining. Mean % CCR2⁺ FEV1/ FEV1% cells per ID# Disease AgeGender Race Smoker FVC pred sample DONOR D1 33 M W N ND ND 1.66 D2 19 MW N ND ND 0.53 D3 23 M W N ND ND 0.19 D4 15 F B N ND ND 2.09 D5 17 M W NND ND 23.97 D6 14 M W N ND ND 1.16 D7 17 M W N ND ND 0.57 D8 52 M W N NDND 0.41 D9 54 M W N ND ND 4.52 D10 19 M W N ND ND 6.52 D11 62 M W N NDND 9.30 n = 11 Median 19 1.66 Range 14-62 0.19-23.97 COPD C1 COPD 58 M WFormer 0.21 17 8.49 C2 COPD 51 M W Former 0.15 14 18.65 C3 COPD 58 F WFormer 0.21 24 9.51 C4 COPD 54 M W Former 0.31 17 9.19 C5 COPD 43 M WFormer 0.44 22 1.61 C6 COPD 61 F W Former 0.36 18 5.35 C7 COPD/A1E 35 FW Former 0.24 17 13.55 C8 COPD 54 F W Former 0.25 16 13.58 C9 COPD 62 MW Former 0.22 18 19.70 C10 COPD 57 F W Former 0.25 16 6.22 C11 COPD 60 FW Former 0.34 20 11.78 C12 COPD 57 F W Former 0.32 30 22.03 C13 COPD 54M W Former 0.17 16 22.25 C14 COPD 63 F W Former 0.28 12 21.71 C15COPD/A1E 52 M W Former 0.27 20 24.11 C16 COPD/A1E 58 F W Former 0.25 1712.09 n = 16 Median 57 0.25 17 12.82 Range 35-63 0.15-0.44 12-301.16-24.11 Abbreviations: M, male; F, female; W, white; B, black; COPD,chronic obstructive lung disease; A1E, Alpha-1-antitrypsin deficiency;FEV1/FVC, ratio of forced expiratory volume at 1 sec to forced vitalcapacity; FEV1% pred, predicted percent FEV1; ND, not determined.

Mice.

The Institutional Animal Care and Use Committee approved these studies.Wild type (n=77) and CCR2-deficient (CCR2^(−/−), n=4) mice (C57BL6/Jstrain) were 8 to 12 weeks of age, of both sexes and approximately 25 gin weight. Mice were used for radiotracer stability studies (n=3) andlung injury studies that included: immunostaining for CCR2 (n=6), invivo immune cell peptide binding (n=18), lung water weight measurements(n=6), in vivo biodistribution (n=12) and PET/CT imaging (n=36). For thelung injury studies, mice were not treated (naive, n=8), administeredintratracheal vehicle control, phosphate buffered saline (PBS, 1 μL/g,n=26) or lipopolysaccharide (LPS, endotoxin, E. coli strain 055:B5,Sigma-Aldrich, St. Louis, Mo.), at a dose of 2.5 μg/g (n=44), “high”dose, 10 μg/g (n=3), or “low” dose, 0.5 μg/g (n=3) LPS. AllCCR2-deficient mice were administered an LPS dose of 2.5 μg/g. Lungwater was quantified using wet-to-dry weight ratios in PBS- orintermediate dose LPS-treated mice (n=3/group). Lungs were resected andweighted immediately, then compared to weights obtained after drying at65° C. for 48 h. The study flow is shown in FIG. 19.

Synthesis, Labeling, and Stability of ECL1i.

The ECL1i peptide (LGTFLKC) was synthesized from D-form amino acids byCPC Scientific (Sunnyvale, Calif.). DOTA-ECL1i was prepared byconjugating 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) that was modified as maleimido-mono-amide-DOTA (1.573 mg, 0.2μmol; Macrocyclics, Dallas, Tex.) to the cysteine residue of ECL1i,using established methods (22). The crude conjugate was purified by highperformance liquid chromatography (HPLC) to reach 99% chemical purityand verified by mass spectrometry. Copper-64 (⁶⁴Cu) was selected as aninitial radiolabel for ECL1i based on a high specific activity thatenabled trace amount administration and provided a decisive PET signalif present, straightforward radiochemistry through the conjugation ofthe DOTA chelator on the peptide, on-site availability, and priorexperience with this radionuclide (22). The DOTA-ECL1i conjugate wasradiolabeled with ⁶⁴CuCl₂ as described (22). Copper-64-DOTA-ECL1i(⁶⁴Cu-DOTA-ECL1i) was tested for stability by incubation in mouse serumat 37° C. for 1 h and in vivo, in blood and lung 1 h post injection(n=3), by radio-HPLC analysis. Maleimide-modified Dylight 550(ThermoFisher Scientific, Waltham, Mass.) was conjugated to ECL1i on thecysteine residue following the same protocol for DOTA conjugation andpurified by HPLC.

Biodistribution.

Mice (n=4/group) were naive, treated with intratracheal PBS or LPS andafter 24 h injected with ⁶⁴Cu-DOTA-ECL1i by tail vein as a bolus of 100μL (3.7 MBq per mouse). After euthanasia, 1 h post-radiotracerinjection, organs of interest were collected, weighed and assayed bygamma counter (Beckman, Brea, Calif.) as described (23). Standards wereprepared and measured in parallel to calculate the percentage of theinjected dose per gram of tissue (% ID/g).

PET/CT Imaging and Image Analysis.

Dynamic (0-60 min) PET and corresponding x-ray computed tomography (CT)images were acquired using cross-calibrated Inveon microPET/CT (Siemens,Malvern, Pa.) or Focus 220 PET (Concorde Microsystems, Knoxville, Tenn.)scanners, immediately after the tail vein injection of ⁶⁴Cu-DOTA-ECL1i(3.7 MBq per mouse) at 24, 48 and 144 h after LPS treatment. The organuptake was calculated as percent injected dose per gram (% ID/g) oftissue in three-dimensional regions of interest (ROIs) from PET imageswithout correction for partial volume effect using Inveon ResearchWorkplace software (Siemens) (23). Time-activity curves were calculatedfrom the ROIs from PET images obtained in a subgroup (n=3/group) of thePBS- and LPS-treated mice undergoing imaging at 24 h. Competitive PETblocking studies were performed in the LPS mouse model with co-injectionof an excess amount of non-radiolabeled ECL1i (900.9 pmol) and⁶⁴Cu-DOTA-ECL1i (1.8 pmol) (ECL1i:⁶⁴Cu-DOTA-ECL1i molar ratio=500:1)followed by a 0-60 minute dynamic scan. PET/CT images of naive (n=4) andPBS-treated (n=5) mice at 24 h were used as controls for allLPS-treatment studies performed at 24 h including ECL1i blocking studies(n=4), CCR2^(−/−) mice (n=4), low- (n=3) and high-dose LPS (n=3), aswell as images obtained at 48 (n=3) and 144 (n=3) h post-LPS.

Flow Cytometry.

Mice (n=4 PBS, n=5 LPS) delivered intratracheal PBS or LPS, and 24 hourslater were injected intravenously with ECL1i labeled with Dylight 550(100 μg) to determine the type of inflammatory cell binding ECL1i. Othermice were treated in parallel but not given ECL1i-Dylight 550 (n=4 PBS,n=5 LPS). One h post-injection, a single cell suspension was producedfrom the lungs, immunostained with immune cell markers (TABLE 2) andanalyzed by flow cytometry.

TABLE 2 Antibodies used to phenotype cells by flow cytometry FluorescentLabel PerCP/Cy5.5 APC Cell Type AF 488 PE/Cy7 AF 647 Neutrophil CD45.2+Ly6G^(Hi) GR-1^(Hi) Monocyte CD45.2+ Ly6G^(Lo) Ly6C^(Hi) MacrophageCD45.2+ CD11c^(Hi) CD11b^(Lo) Dendritic Cell CD45.2+ CD11c^(Hi)CD11b^(Hi) T Cell CD45.2+ — CD90.2 (Thy 1.2)^(Hi) B Cell CD45.2+ —CD19^(Hi)

Immunostaining and Microscopy.

Tissue sections were immunostained using a CCR2 monoclonal antibody(Novus Biologicals, Littleton, Colo.) in mouse and human tissue (n=6mice, n=27 human). In each human tissue section, CCR2-staining cellswere assessed from five photomicrographs, acquired at 200×magnification. Images were analyzed by investigators who were unfamiliarwith the hypothesis. The percent of CCR2 expression was determined as aratio of the area of CCR2 signal relative to DAPI using ImageJ softwarev1.50. Details are provided in the Supplement.

Autoradiography.

Fixed human lung tissue sections that were deparaffinized and hydratedin PBS were incubated with ⁶⁴Cu-DOTA-ECL1i for 5 min (n=4 donor, n=6COPD). Blocking studies were performed in lung sections from subjectswith COPD demonstrating levels of CCR2 immunostaining above the median(n=6) by co-incubation with 500-fold non-radioactive ECL1i. Slides werewashed 30 times with water then placed in an instant imager (Packard,Meriden, Conn.) for 30 minutes. Images were post-processed using ImagerSoftware (Packard, Meriden, Conn.) and the percentage of blocked signalwas calculated.

Statistical Methods.

Data were analyzed using Prism (version 6.07, Graphpad, La Jolla,Calif.). Differences between groups were compared using the two-tailedStudent's t-test. Means of non-parametric data from human samples wascompared using the Mann-Whitney U test. Multiple means were comparedusing a one- or two-way ANOVA with Tukey's test. Significance wasestablished as p<0.05.

⁶⁴Cu-DOTA-ECL1i In Vivo Stability Study.

⁶⁴Cu-DOTA-ECL1i (3.7 MBq in 100 μL saline) was injected into the tailvein of mice. At 1 h post injection, mice were euthanized and blood wascollected in a glass tube containing acid citrate dextrose. The plasmawas separated from cells by centrifugation then analyzed by HPLC(Ultimate 3000, Dionex, Sunnyvale, Calif.). The HLPC instrument that wasequipped with a UV/VIS detector (Dionex, Sunnyvale, Calif.), aradioactivity detector (B-FC-3200; BioScan Inc., Poway, Calif.) and aC-18 column (5 mm, 4.6×220 mm; Perkin Elmer, Waltham, Mass.) (36).Similarly, mouse lung was harvested one hour after radiotracer injectionand rinsed with PBS (5 mL, three times) prior to cell disruption using aprobe sonicator (Sonifier 185 cell disruptor, Branson, Danbury, Conn.)for 30 seconds. The supernatant was isolated by centrifugation andanalyzed by HPLC. The eluate was separated by 1 mL fractions and countedin a well gamma counter (Wallac Wizard 1470, Perkin Elmer, Waltham,Mass.).

Mouse Lung Injury Model.

Wild-type mice in the C57B/6J background and CCR2-deficient mice(CCR2^(−/−), No. 004999) were obtained from the Jackson Labs (BarHarbor, Me.). CCR2-deficient mice were backcrossed into the C57B/6Jbackground. Mice, 8 to 12 weeks of age, of both sexes and weighingapproximately 25 g, were used. Mice were anesthetized by administering acombination of ketamine HCl, 100 mg/kg and xylazine HCl. 15 mg/kg, byintraperitoneal injection. To deliver PBS control vehicle or LPS intothe lung, the trachea was surgically isolated and cannulated with a 22gauge, 1″ catheter (Exel Safelet, ThermoFisher Scientific, Waltham,Mass.). Mice were administered phosphate buffered saline (PBS, 1 μL/g)or lipopolysaccharide at a dose of 2.5 μL/g (LPS, endotoxin, E. colistrain 055:B5, Sigma-Aldrich, St. Louis, Mo.). Other mice wereadministered an LPS “high” dose, 10 μL/g, or “low” dose, 0.5 μg/g, usingstock solutions of LPS created to inject a volume of 1 L/g.Intratracheal injection was over approximately 2 seconds, after whichmice were kept warm and allowed to recover.

Flow Cytometry.

Mice delivered intratracheal PBS or LPS were injected intravenously withECL1i labeled with Dylight 550, as with the radiolabeled probe, todetermine the type of inflammatory cell binding ECL1i. For eachexperiment, animals treated with PBS or LPS were paired with a controlnot injected with ECL1i-Dylight 550 to determine the background signal.Lungs were removed from mice without flushing blood from thevasculature, minced on ice and digested in Roswell Park MemorialInstitute (RPMI) 1640 medium containing Liberase TL 50 μg/mL (Roche,Indianapolis, Ind.) and deoxyribonuclease (DNAse) I, Type II, 20 U/mL(Sigma-Aldrich) at 37° C. for 60 min. The digested tissue was passedthrough a 70-μm sieve (Falcon 352350, Corning, Corning, N.Y.) to createa single cell suspension, and then incubated in ACK Lysing Buffer(Lonza, Walkersville, Md.) at room temperature to lyse red blood cells.After 5 minutes the sample was neutralized with FACS buffer (PBS with 2%fetal bovine serum) and centrifuged at 500 g for 8 minutes at 4° C.Cells were suspended in FACS buffer, counted by hemocytometer, andprepared for flow cytometry. Cells were immunostained with labeledantibodies (all from BioLegend, San Diego, Calif., unless indicated)specific for mouse, including CD31 PerCP/Cy5.5, CD326 Alexa Fluor 488,CD45.2 Alexa Fluor 488, Gr-1 APC (eBioscience), Ly-6C APC, CD11c PE/Cy7,CD11b APC, CD90.2 (Thy-1.2) APC, Ly-6G PE/Cy7 (BD Pharmingen), and CD19APC. Counts from 1 to 10×10⁴ cells were collected for each sample on aFACSCalibur (Becton Dickinson), dual laser, flow cytometer usingCellQuest Pro software (BD Biosciences), and analyzed using FlowJosoftware (Ashland, Oreg.). Cell phenotype was determined by antibodybinding shown in the TABLE 2. The mean fluorescent intensity wasdetermined by calculating the geometric mean of Dylight 550 fluorescenceof the cell types identified in ECL1i-Dylight 550-injected mice andsubtracting the fluorescent background of the same cell types fromsimilarly treated mice not injected with ECL1i-Dylight 550.

Immunostaining and Microscopy.

Tissue sections were fixed and processed for immunostaining as described(37). The monoclonal anti-CCR2 antibody (E68, Novus Biologicals,Littleton, Colo.) was used in mouse and human tissues. In mouse lung,the CCR2 antibody was detected using avidin-biotin amplification(Vectastain Elite ABC, Vector Laboratories, Burlingame, Calif.) andhorseradish peroxidase (HRP) substrate 3, 3-diaminobenzidine (DAB),which produces a brown reaction product. Tissues were thencounterstained blue with hematoxylin. In human tissues, the CCR2antibody was detected with an Alexa Fluor 555 labeled secondary antibody(Molecular Probes, Carlsbad Calif.) and DNA was counterstained with 4′,6diamidino-2-phenylindole (DAPI). Photomicroscopy was performed using aLeica DM5000 microscope and DFC7000T camera interfaced with LASXsoftware (Leica Microscopy, Buffalo Grove, Ill.). Images were adjustedglobally using Photoshop software (Adobe, San Jose, Calif.). In humantissue CCR2-staining cells were assayed from five representativephotomicrographs from each tissue section. Images were acquired at 200×magnification and counted by investigators with expertise in cellbiology blinded to the hypothesis (ZBN, 2 years of experience; JP, 25years of experience, and SPG, 9 years of experience) that results aredifferent between study groups and with experience in scoring thesedata. The CCR2 expression was calculated as a ratio of the area of CCR2signal relative to DAPI determined using ImageJ software v1.50 toestablish a threshold setting for positive staining cells (38). Oncedetermined, values from the five representative images were from allsamples were collected, analyzed by threshold, then averaged, all by asingle investigator (ZBN). The percent of CCR2 positive cells in thedonor and COPD groups were compared using the Mann-Whitney test. Tofurther assess our findings, we also immunostained additional lungsamples from different regions of the lung that were available for someindividuals (n=2 donors, n=3 COPD). Typically, there was littlevariation in the level of CCR2⁺ cells within the same specimen. In thesecases, the mean percent CCR2⁺ cell value was calculated and used foranalysis.

⁶⁴Cu-DOTA-ECL1i Radiochemistry and Stability.

The mass spectrometry of the conjugate confirmed that one DOTAconjugated to one ECL1i peptide (M⁺ calculated: 1306.65, observed:1306.69) (see e.g., FIG. 20A). The radiochemical purity of⁶⁴Cu-DOTA-ECL1i used for animal studies was 98% or greater, confirmed byradio-HPLC (see e.g., FIG. 20B). The specific activity of⁶⁴Cu-DOTA-ECL1i was 55.5±1.11 mCi/nmol (n=20), enabling trace amounts(˜70 pmol) to be injected for in vivo studies. ⁶⁴Cu-DOTA-ECL1i wasstable in mouse serum in vitro at 37° C. for 1 h (98.2±2.1%) and invivo, in serum (96.5±1.1%) and lung (95.6±3.0%) recovered from naivemice 1 h post injection, using HPLC analyses and gamma counting (seee.g., FIG. 21).

Biodistribution of ⁶⁴Cu-DOTA-ECL1i in the LPS Mouse Lung Injury Model.

We assayed ⁶⁴Cu-DOTA-ECL1i in a well-characterized lung injury model inwhich endotoxin (LPS) activates the accumulation of CCR2-expressingcells in the lung (13, 24). As expected, 24 h after intratrachealdelivery of LPS, CCR2-expressing cells were detected in mouse lungs (seee.g., FIG. 22A). The in vivo pharmacokinetic evaluation of⁶⁴Cu-DOTA-ECL1i acquired 1 h following intravenous injection wascompared in naive mice and 24 h after the delivery of intratrachealcontrol vehicle PBS or LPS (see e.g., FIG. 22B). Accumulation of tracerin the lung of mice given LPS was 2.5 times higher than that in the PBSgroup, although there was no difference in blood retention.⁶⁴Cu-DOTA-ECL1i showed renal clearance as evidenced by the kidneyaccumulation in both groups. Liver and bone marrow uptake of⁶⁴Cu-DOTA-ECL1i in LPS-treated mice was significantly higher than thePBS-treated mice.

CCR2 Imaging in LPS Lung Injury.

Based on the biodistribution studies, we administered intratracheal PBSor LPS and determined if intravenous ⁶⁴Cu-DOTA-ECL1i could be used as aPET agent to image lung inflammation. Compared to PBS delivery, therewas a prominent signal in the lungs of LPS-treated mice at 24 h (PBSmean % ID/g=0.99±0.29, n=5; LPS mean % ID/g=4.43±1.44, n=7; P<0.001)(see e.g., FIG. 23). Consistent with biodistribution studies, LPS alsoincreased activity in the liver, and renal clearance was indicated byenhanced kidney and bladder activity in all mice. The time-activitycurve acquired 24 h after injury demonstrated a higher and consistentaccumulation of ⁶⁴Cu-DOTA-ECL1i in the LPS-treated lung compared to thePBS control, which was significantly lower, and further diminished overthe imaging period. ⁶⁴Cu-DOTA-ECL1i uptake in the lung peaked at 24 h,followed by a loss of signal in mice injected and imaged at 48 h (mean %ID/g=1.04±0.17, n=5) or 144 h (mean % ID/g=1.29±0.32, n=5) post-LPS (seee.g., FIG. 23C). Mice were administered high and low doses of LPS todetermine the sensitivity of ⁶⁴Cu-DOTA-ECL1i detection by PET. The lungsignal in mice administered low dose LPS (mean % ID/g=1.82±0.17, n=5)was decreased compared to treatment with intermediate and high doses(see e.g., FIG. 23D).

The specificity of the ECL1i radiotracer was examined in additionalcontrol conditions (see e.g., FIG. 24). The level of ⁶⁴Cu-DOTA-ECL1ilung signal in naive mice (mean % ID/g=0.39, n=3) was negligible and notsignificantly different from that observed in mice treated with PBS.Moreover, the signal in these conditions was similar to LPS-treated micethat were co-injected with excess non-radioactive ECL1i plus⁶⁴Cu-DOTA-ECL1i (blocked) (mean % ID/g=0.63±0.15, n=4, P<0.001). Therewas also nearly complete loss of lung signal in LPS-treatedCCR2-deficient mice (mean % ID/g=0.39±0.04, n=3, P<0.001). We consideredthat blood flow may contribute to differences in the PET signal betweenLPS-treated and control mice, however, there were not significantdifferences in lung weight wet-to-dry ratios in PBS-compared toLPS-treated mice (5.20±0.20 g vs. 5.34±0.34 g, n=3/group, P=0.650).

ECL1i Tracer Binds Monocytes In Vivo.

To determine the cell types that bound ECL1i in wild type mice, thepeptide was tagged with the fluorescent dye Dylight 550. At 24 hpost-intratracheal PBS or LPS delivery, ECL1i-Dylight 550 was injectedintravenously and one hour later, lungs were analyzed by flow cytometry.LPS induced significant binding of ECL1i-Dylight 550 to monocytes(Ly6G^(lo), Ly6C^(hi)) (see e.g., FIG. 25). ECL1i-Dylight 550 also boundlung to macrophages (CD11b^(hi), CD11c^(low)) and a very small group of“bright” dendritic cells (CD11b^(hi), CD11c^(hi)), consistent with knownCCR2⁺ populations (1, 2).

CCR2 Detection in Lung Tissue from Subjects with COPD.

The percentage of CCR2⁺ cells was significantly increased in lungtissues from COPD subjects (median 12.82 percent cells/sample, range1.16-24.11) compared to lung donors (median 1.66 percent cells/samplerange 0.19-23.67; P=0.002) (see e.g., FIG. 26, TABLE 1). However, therewere a wide range of CCR2⁺ cells, with the levels being similar intissues from some COPD and non-COPD subjects.

Accordingly, we next tested the binding of ⁶⁴Cu-DOTA-ECL1i in the lungtissue sections from COPD and donor subjects using autoradiography onslides. To enhance the differences in CCR2 detection, we studied sampleswith high versus low numbers of CCR2⁺ cells detected by immunostaining.This comparison showed that ⁶⁴Cu-DOTA-ECL1i binding was increased inrepresentative samples with high numbers compared to those with lownumbers of CCR2⁺ cells (see e.g., FIG. 26C). Qualitative assessment ofprobe binding was determined by blocking the ⁶⁴Cu-DOTA-ECL1i signalusing non-labeled ECL1i in tissue samples from COPD subjects with highlevels of CCR2⁺ cells. In all samples tested (n=6), competition withnon-labeled ECL1i diminished the level of ⁶⁴Cu-DOTA-ECL1i binding basedon autoradiographic signal (see e.g., FIG. 26D).

DISCUSSION

We selected a CCR2 binding peptide as a PET imaging target based onseveral observations. First, the CCR2/CCL2 axis recruits inflammatorymonocytes and other types of immune cells into the lung (1). Second,CCR2 is elevated in lung cells in ARDS, COPD, experimental asthma andpulmonary fibrosis, as well as common non-pulmonary diseases, includingatherosclerosis and malignancy (6, 8-10, 17, 25-28). Third, there issubstantial evidence that genetic deletion or pharmacologic inhibitionof CCR2 ameliorates disease in animal models leading to industry effortsto develop and trial CCR2 antagonists for respiratory disorders andother disease (2, 18, 19).

Current tools are limited for the non-invasive assessment ofinflammation in lung disease. To test ⁶⁴Cu-DOTA-ECL1i as an agent todetect inflammation, we chose LPS injury, a well-established model thathas been characterized relative to CCR2-mediated inflammation (13, 24,29). Prior reports demonstrate that LPS directs accumulation ofCCR2-expressing cells in the lung in wild type, but not CCR2^(−/−) mice,or after anti-CCR2 antibody blockade (13, 24, 30). Consistent with thesefindings, ⁶⁴Cu-DOTA-ECL1i activity in the lung was increased only duringthe acute phase of injury and was not present in CCR2^(−/−) mice. Thespecificity of ⁶⁴Cu-DOTA-ECL1i activity was further shown by the abilityto block detection of LPS-induced activity using non-radioactive ECL1i.

Because CCR2 deletion in mice alters immune responses, includingdecreased neutrophil influx and other inflammatory cells in the lung(13-15), we sought alternative approaches to study the in vivoperformance of ECL1i. To determine the cell types binding ECL1i wecreated a fluorescently tagged ECL1i imaging agent for in vivo labelingof immune cells in wild type mice. Injection of ECL1i-Dylight 550 andanalysis of whole lung cell preparations by flow cytometry revealedECL1i signal in lung monocytes and macrophages, as well as in smallnumbers of dendritic cells, also known to express CCR2 (12, 26, 29).Thus, future in vivo studies using ECL1i-Dylight 550 with CCR2 reportermice may provide additional information as to cell targets. Ultimately,characterizing the complete identity of cell types that bind⁶⁴Cu-DOTA-ECL1i in vivo will be difficult owing to inherent differencesin the sensitivity of detection methods for fluorescent and radionuclidelabels.

⁶⁴Cu-DOTA-ECL1i joins a small number of agents developed for PET imagingof non-malignant lung disease. The strength of the ECL1i radiotracer isan ability to image CCR2-related inflammation, as we have shown in thelung using the mouse endotoxin and a mouse ischemia-reperfusion model oflung transplantation (21). ⁶⁴Cu-DOTA-ECL1i activity in the bone marrowand extrapulmonary organs suggests that it may also be possible tofollow CCR2 cell trafficking. Moving forward, It will be important tocompare the utility of ⁶⁴Cu-DOTA-ECL1i with radiotracers that are lessspecific for a defined immune cell population, such as ¹⁸FDG PET (5,31),and using single photon emission computed tomography or planar imagingwith technetium-99m hexamethylpropylene amine oxime (^(99m)Tc-HMPAO)(32), as well as targeted approaches including the translocator protein(TSPO) (33) and folate receptor 3 (34) to identify CCR2-dependentinflammation.

We identified that the percent of CCR2 expressing cells detected byimmunostaining was elevated in lung tissue from subjects with severeCOPD compared to the lung donor group. This finding was consistent withprior reports of elevated levels of CCR2⁺ cells in other types ofsamples obtained from individuals with COPD (6,7). There was someoverlap in levels between the COPD and donor groups, possibly due tounderlying phenotypic differences in COPD and inflammation that mayoccur in the donor lungs prior to harvest for transplantation. Thus,further study will be needed to define precise differences for CCR2⁺cells in COPD, but the present observations identify a set of clinicalsamples and a strategy that can be used to validate whether the level ofCCR2⁺ cells in tissue correlates with the level detected using our probefor non-invasive imaging.

We have assessed radiotracer activity in a limited number of mice and⁶⁴Cu-DOTA-ECL1i has a very rapid blood clearance (<1% ID/g at 1 h postinjection), so that the sensitivity of detection may be limited.Chemical modification of the radiotracer could improve thepharmacokinetics to increase the blood retention time. While we haveshown that ECL1i can bind human tissues in from subjects with COPD,sampling issues inherent in this heterogeneous disease preventgeneralization to the whole lung. Assessing mice was performed on arelatively small numbers of have been studied with PET/CT following⁶⁴Cu-DOTA-ECL1i injection and models of chronic lung disease such asCOPD are lacking; cigarette smoke in mice does not induce inflammationsimilar to that in humans (35). Interpretation of results is alsolimited by a lack of current approval for clinical testing. Moving tohuman trials may be the only way to test our proposal that a⁶⁴Cu-DOTA-ECL1i signal is increase in the lungs of subjects with COPD.While the ⁶⁴Cu decay half-life of ˜13 h may be less desirable forimaging, the high specific activity and the longer physical half-lifewill permit production and distribution of intact radiotracer fornationwide trials. For expanded clinical use of ECL1i, radiochemistrycould be developed for flouride-18 labeling. Moreover, future humanstudies of PET imaging using radiolabeled ECL1i in combination withcomputed tomography x-ray may provide synergistic information regardingthe localization of inflammation, and the relationship to specific lungstructures and pathologic changes.

Practical Applications.

A CCR2 binding peptide adapted as a PET probe can detect lunginflammation in a mouse model and human tissues, and may serve as a toolfor the management of human lung disease. Our study of lungs of subjectswith clinically similar, very severe COPD showed that expression of CCR2varied markedly among subjects, supporting the concept that themolecular mechanisms underlying chronic inflammatory lung pathologiesare not necessarily alike, but could be differentiated by non-invasivedetection of certain biomarkers. There are few available treatments forCOPD, and CCR2 detection could be an important step for developingtherapies, personalizing treatment and monitoring treatment response.

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Example 3: Imaging Mouse Models of Lung Diseases, Disorders, andConditions

The following example describes methods of imaging CCR2 receptors in thelung to guide diagnosis and therapy. Lung diseases are oftencharacterized by the nature of immune or inflammatory cells that arefound within the tissues and airways¹⁻⁴. Chemokines guide the migrationand function of inflammatory cells harboring their cognatereceptor^(5,6). In the lung, the chemokine CCL2 (monocytechemoattractant protein-1, MCP-1), is frequently elevated in acute andchronic lung disease⁷⁻⁹. CCL2 is the major ligand for the chemokinereceptor CCR2, which is found largely on immune cells and notably onmonocytes, dendritic cells (DCs) and T cells^(5,6,10-12).

Why Image CCR2⁺ Cells in Acute and Chronic Lung Disease?

CCR2 was selected as a PET imaging target based on: (1) knowledge thatthe CCR2/CCL2 axis recruits inflammatory monocytes and other cell typesinto the lung (and other organs); (2) elevation of CCR2/CCL2 in acuteand chronic lung diseases; (3) evidence that genetic deletion orpharmacologic inhibition of CCR2 ameliorates disease in animal models;(4) industry efforts to develop and trial CCR2 antagonists for lung andother disease (e.g., diabetes, malignancies); and (5) an inability tonon-invasively identify CCR2⁺ cell burden in tissues for optimalpersonalized treatment.

CCR2 Directs Monocytes and Other Immune Cell Recruitment in the Lung.

A major role for the CCL2/CCR2 pair is the recruitment of inflammatorymonocytes from the bone marrow^(7,13) and regulation of macrophage,dendritic and T cells maturation^(9,14). In response to CCL2, CCR2⁺monocytes adhere to the vascular endothelial surface and migrate intotissue, along chemotactic gradients¹⁵. Inflammatory monocytes (mouseLy6C^(hi) Ly6G^(lo), human CD14⁺CD16⁻) serve as precursors for classicalmacrophages and conventional DCs¹⁶. CCR2⁺ monocytes also provide asecondary source of proinflammatory modulators, such as tumor necrosisfactor-α, interleukin-1β and matrix metalloproteinases, contributing tolung injury¹⁷⁻¹⁹. Although inflammatory monocytes are essential earlyresponders, excessive or prolonged recruitment impairs resolution ofinflammation and propagates disease progression.

CCL2/CCR2 is Elevated in Lung Disease.

As described herein, CCR2 can act as a biomarker for lung inflammationused to stratify treatments (e.g., anti-inflammatory or CCR2antagonists) and to monitor disease. The CCL2/CCR2 axis is demonstratedto be active in acute and chronic lung diseases. CCR2 function issupported by deletion or antagonism in related mouse models. Diseasesinclude those for which specific therapies are limited, including thosedetailed below.

Acute Lung Injury.

Excessive recruitment of CCR2-dependent leukocytes impacts thepathogenesis of acute lung injury in human ARDS and mouse models,shaping the magnitude and duration of disease²⁰⁻²². Endotoxin (LPS)triggers CCR2-depedent migration of monocytes to the lung whenadministered intratracheally and also influences subsequent neutrophilrecruitment in lung.

PGD.

Reperfusion injury immediately following lung transplant, known asprimary graft dysfunction (PGD), is marked by elevated CCL2 levels inBAL fluid, while clinical improvement occurs as CCL2 levels fall. It wasobserved that CCR2 is required for mobilization of CD11b⁺Ly6C^(hi)monocytes and accumulation into lung allographs in a mouse lungtransplant model of PGD.

COPD.

Human studies of CCL2/CCR2 in COPD show increased levels of CCL2 in thesputum, BAL fluid and lungs (including ex-smokers) and expression ofCCR2 on leukocytes and epithelia. Our group recently reported increasedCCR2 on interstitial monocytes from COPD lung tissue.

Asthma.

In human subjects with asthma studied by segmental allergybronchoprovocation, CCL2 and CCR2 were increased in BAL. Blocking orgenetic deletion of CCL2/CCR2 in mouse models of airway allergensensitization prevents monocyte and dendritic cell migration andallergic responses^(8,36-38).

Pulmonary Fibrosis and Others.

Human and mouse studies have also implicated the CCL2/CCR2 axis in thepathogenesis of pulmonary fibrosis, bronchiolitis obliterans syndrome,and fungal pneumonia.

CCR2 Plays Roles in a Broad Number of Pathologic Processes Beyond theLung.

The importance of CCR2 as a biomarker and as a radiotracer extends wellbeyond the lung. Experimental evidence for CCR2 deletion includesattenuation of atherosclerotic plaques, myocardial infarction, sepsis,allograph rejection, glucose control and nephropathy in diabetes,symptoms in multiple sclerosis and the extent of tumor metastasis.

Clinical Trials of CCR2 Antagonists.

A remarkable number of CCR2 antagonists have been produced and thewebsite ClinicalTrials.gov lists 16 CCR2 antagonist trials. Smallmolecules are in trials for atherosclerosis, rheumatoid arthritis,multiple sclerosis, pancreatic cancer and other diseases. In one study,CCR2 antagonists decreased urinary albumin and improved glycemiccontrol. As for lung disease, a Phase 1 study of COPD in Eastern Europereported no toxicity (NCT01215279). Other trials of antagonists havefailed, possibly due to an inability to molecularly phenotype subjectsand monitor a CCR2-specific endpoint.

CCR2 PET imaging can serve to phenotype, monitor, and guide treatment.

Chemokine receptor phenotyping is a step toward precision medicine. Lungmedicine lags considerably behind oncology and other disciplines wherethe use of molecular markers has become the standard of care. Theinability to easily molecularly phenotype patient populations is asignificant gap in the field and has possibly slowed progress.Non-invasive chemokine receptor phenotyping technology using PET imagingcan drive the field. Inflammatory cells cannot be accurately sampledfrom the lung. Bronchoalveolar lavage (BAL) is restricted to those cellswithin the airspaces rather than the interstitial spaces, which is aresidence for inflammatory populations. Biopsy is more invasive andsuffers from sampling error. Given the high sensitivity, quantification,and translation capability of PET, as documented for oncologicalapplications, CCR2 PET imaging can be rapidly adopted for translationalresearch. Thus, a robust and accurate non-invasive method to measureCCR2 expression is highly attractive from a disease staging and drugdiscovery perspective.

Novel Chemokine Receptor Radiotracer Development.

CCR2 targeting ligand (ECL1i) and radiotracer (⁶⁴Cu-DOTA-ECL1i) arenovel. Our group has been a leader in the preclinical development of PETimaging of chemokine receptors—we developed a pan-chemokine receptor(vMIP-II) and CCR5-based tracers in mouse models of vascularinflammatory lesions. CXCR4 is the sole other chemokine receptoridentified by PET imaging. A ⁶⁸Ga-CXCR4 antagonist was recently used forimaging human cancer.

Broadly Applicable CCR2 Radiotracer.

As there are no imaging techniques for the non-invasive detection ofCCR2, the radiotracer described in this project, if successful, willprovide a valuable tool for studying pro-inflammatory immune celltrafficking in lung and other disease conditions. A CCR2 radiotracer mayalso permit the characterization of monocyte mobilization from bonemarrow and spleen. Such information would provide the foundation forpotential paradigm shifting approaches to the diagnosis and treatment ofseveral common human diseases.

Lung Injury Model Development.

Together, we have established lung injury models (e.g., lungtransplantation, transgenic mice) for the optimal testing of radiotracersensitivity and specificity and a unique biorepository of phenotypedhuman lung tissues and cells from subjects with advanced lung disease totest our probe.

Approach.

The data provided herein describes the development and characterizationof a CCR2 binding peptide, ECL1i designed with ⁶⁴Cu radiolabeling(⁶⁴Cu-DOTA-ECL1i) for PET imagining. ⁶⁴CU was chosen as a widely usedPET radioisotope, regularly produced by the Washington Univ. CyclotronFacility, with desirable nuclear properties (t_(1/2)=12.7 h, β⁺=0.653Mev (17.8%), β⁻=0.579 Mev (38.4%)) for pre-clinical and clinicalinvestigation. Data includes tests of ⁶⁴Cu-DOTA-ECL1i specificity,sensitivity and safety in several different models to best validateradiotracer function. Moving this agent to human safety studies, bymeans as described below, to validate the radiotracer in mice, can beperformed by confirming binding in human cells, gaining FDA approval,and performing Phase 0 safety studies. The best mouse models to validatetracer performance will be used, making no attempt to “model” a humanlung disease for which no mouse model is truly authentic and defensible.However, there is sufficient new and established CCR2-related human datato direct thee studies without mouse models. The multidisciplinary teamcan generate the preclinical data and perform Phase 0/Early Phase 1studies. Once accomplished, ⁶⁴Cu-DOTA-ECL1i will be tested in Phase 1studies for safety in a targeted population. Below are data that supportthe envisioned human studies.

Development of PET Chemokine Receptor Lung Imaging.

To determine if a radionuclide-labeled chemokine receptor-bindingpeptide could be used to detect lung inflammation, we employed the68-residue vMIP-II derived from human Herpes virus 880. vMIP-II bindsand antagonizes activity of chemokine receptors CCR1 through 5 and 8,CXCR3 and 4, CR1, CX3CR1 and XCR1^(80,81). We hypothesized that vMIP-IIcould serve as a broad-spectrum probe for lung inflammation. The Liu labshowed that PET imaging using i.v. ⁶⁴Cu-DOTA-vMIP-II detected chronicinflammation following vascular injury in ApoE^(−/−) mice⁸².Accordingly, C57BL/6 mice administered intratracheal PBS or endotoxin(LPS, E. coli 055:B5, 2.5 mg/mL/kg)⁸³ were injected with⁶⁴Cu-DOTA-vMIP-II by tail vein and underwent dynamic PET/CT scans over30 min (see e.g., FIG. 13A). Compared to PBS control, LPS induced a3-4-fold higher vMIP-II signal in the lungs that peaked at 24 h (seee.g., FIG. 13B). When injected with the radiotracer at 48 h, 72 or 144h, the lung signal was similar to control conditions. Concurrentadministration of 100-fold excessive mass of non-radiolabeled vMIP-IIblocked signal at 24 h post-LPS, suggesting that the ⁶⁴Cu-DOTA-vMIP-IIsignal was specific. Moreover, H₂ ¹⁵O and C¹⁵O did not identifydifferences in blood pool at 24 h. Uptake was also observed in the liverin the mice given LPS (likely a systemic inflammatory response⁸⁴), aswell as kidneys and bladder activity, reflecting renal clearance.

vMIP-II Binds Lung Inflammatory Monocytes and Other Cell Types.

To determine the phenotype of cells that bound vMIP-II, fluorescentlytagged peptide (with Dylight 550) was used in a protocol that paralleledthe radiotracer studies. The lungs were digested and analyzed for immunecell markers that co-localized with DOTA-vMIP-II-Dylight550 using flowcytometry. LPS treated mice had vMIP-II⁺ endothelial cells (CD31⁺),monocytes (Ly6G^(lo), Ly6C^(hi)) and a DC population (CD11b^(hi),CD11c^(hi)) at 24 but not 48 h (see e.g., FIG. 13C), consistent with theacute influx of inflammatory monocytes post-LPS^(25,42,85). Two-photonmicroscopy using the same probe identified uptake in subepithelialregions of airways and in alveolar capillaries (data not shown). Toimage a molecularly defined inflammatory monocyte population, weidentified a peptide that bound CCR2.

Identification of a CCR2 Binding Peptide.

ECL1i is a CCR2 binding peptide and antagonist developed by collaboratorC. Combadiere (INSERM, Paris, FR, see letter) based on prior knowledgethat mutagenesis of CCR2 threonine 117 impairs cell migration, but notsignaling⁸⁶. The heptapeptide LGTKLKC, (C) inverso (ECL1i) was designedto correspond and bind to an inverted sequence in the thirdtransmembrane domain at the first extracellular loop (ECL1) of CCR2. Theregion is highly conserved in mouse and human.

Specificity of the CCR2 Binding Peptide ECL1i.

CCR2⁺ cells migrate in response to CCL2. ECL1i specifically inhibitedCCL2-induced chemotaxis in CHO cells that stably express human CCR2(hCCR2) in a concentration-dependent manner and similar to CCR2 theantagonist BMS22^(54,55). However, ECL1i did not induce the migration ofCHO-hCCR2 cells at concentrations that inhibited CCL2 signaling (datanot shown). ECL1i also inhibited CCL2-mediated chemotaxis of CCR2⁺inflammatory monocytes, but not of resident monocytes. ECL1i did notinhibit the migration of cells expressing human CCR1, CCR5 or CX3CR1(not shown). These data indicate that ECL1i selectively inhibitsCCL2-induced chemotaxis of CCR2⁺ cells without inducing chemotaxis ofthese cells. Also, incubation of CCR2-expressing HEK293 cells with ECL1idid not prevent the binding of fluorescently-labeled CCL1 (not shown),indicating distinct sites of interaction.

Synthesis and Characterization of ⁶⁴Cu-DOTA-ECL1i.

We prepared DOTA-ECL1i by conjugating maleimido-mono-amide-DOTA with theCCR2 binding peptide LGTFLKC using established methods⁶⁹. The crudeconjugate was purified by HPLC to reach 99% chemical purity and verifiedby mass spectrometry (M⁺ calculated: 1306.65, observed: 1306.69) (seee.g., FIG. 2A, FIG. 2B). The DOTA-ECL1i conjugate was radiolabeled with⁶⁴CuCl₂ as described⁶⁹ with specific activity of 1.5±0.3 mCi/nmol(n=20), enabling injection of trace amounts (˜70 pmol) for PET imaging.⁶⁴Cu-DOTA-ECL1i was 100% stable after incubation with mouse serum at 37°C. and in vivo, in blood and lung 1 h post injection, by HPLC analysis.

In Vivo Toxicity Studies.

Cytotoxicity assays of non-radioactive Cu-DOTA-ECL1i performed in humanand mouse cells lines by MTT and ATP activity showed no change inactivity compared to controls at concentrations up to 40 μg/mL/10⁵cells. Toxicity studies were performed by S. Greco, DVM (Dept. ofComparative Med., Washington Univ.). Mice given i.v. Cu-DOTA-ECL1i (110μg) at 500-fold the estimated dose for human imaging evaluated 24 hlater showed normal serum hepatic and renal chemistries, and histologicexamination of lung, liver and kidneys (n=5 mice).

CCR2 Imaging in LPS Lung Injury (See e.g., FIG. 14).

CCR2 directs LPS-induced accumulation of monocytes in the lung in adose- and time-dependent pattern in wild type but not CCR2^(−/−) mice orafter anti-CCR2 antibody blockade^(25,85). Thus mice were administeredintratracheal PBS or LPS to determine if ⁶⁴Cu-DOTA-ECL1i could be usedas a PET agent to detect CCR2-expressing cells, as observed byimmunostaining (see e.g., FIG. 14A, FIG. 14B). At 24 h, mice injectedwith ⁶⁴Cu-DOTA-ECL1i via tail vein were imaged by PET/CT for 60 min (seee.g., FIG. 14C, FIG. 14D). Compared to PBS treatment, there was aprominent signal in the lungs of LPS treated mice. ⁶⁴Cu-DOTA-ECL1iuptake in the lung peaked at 24 h, followed by a loss of signal in miceinjected at 48 h or 166 h post-LPS (see e.g., FIG. 14E). Miceadministered a low dose of LPS had a diminished lung signal, suggestingsensitivity (see e.g., FIG. 14D). Loss of lung signal in LPS-treatedCCR2^(−/−) or LPS-treated mice co-inject with excess non-radioactiveplus ⁶⁴Cu-DOTA-ECL1i suggested radiotracer specificity (see e.g., FIG.14F). LPS also increased activity in the liver, consistent with asystemic inflammatory response⁸⁴. Renal clearance was indicated byenhanced kidney and bladder activity in all mice.

Biodistribution of ⁶⁴Cu-DOTA-ECL1i in the LPS Mouse Lung Injury Model.

The in vivo pharmacokinetic evaluation of ⁶⁴Cu-DOTA-ECL1i at 1 hpost-injection was compared in mice 24 h after delivery of intratrachealPBS or LPS (see e.g., FIG. 14G). Following i.v. delivery,⁶⁴Cu-DOTA-ECL1i showed fast renal clearance evidenced by the kidneyaccumulation in both groups. Although there was no difference in bloodretention between LPS and PBS treated mice, the lung accumulation oftracer in LPS group was 2.5 times higher than that acquired in the PBSgroup. As expected, liver, and bone marrow uptake of ⁶⁴Cu-DOTA-ECL1i inLPS treated mice was higher than those in the PBS mice due to the LPSinduced inflammation.

ECL1i Tracer Binds Inflammatory Monocytes In Vivo.

To determine the LPS-induced cell types that bound ⁶⁴Cu-DOTA-ECL1i,ECL1i was tagged with fluorescent Dylight 550 and injected in PBS or LPStreated mice. Cells from lung digest were analyzed by flow cytometry at24 h. LPS induced significant binding of ECL1i-Dylight 550 to monocytes(Ly6G^(lo), Ly6C^(hi)) (see e.g., FIG. 15). A very small population of“bright” DCs (CD11b^(hi), CD11c^(hi)) bound ECL1i-Dylight 550, but wasnot significantly different between conditions. More detailed in vivostudies of ECL1i binding population are described in Example 3A.

CCR2 Imaging in Acute Lung Reperfusion Injury (See e.g., FIG. 6A).

Primary graft dysfunction PGD) is a significant cause of post lungtransplant morbidity and is associated elevated CCL2 levels in BALfluid^(27,28). PGD presents as respiratory failure immediatelypost-transplant, posing a broad differential diagnosis. Current therapyis non-specific—to intensify immunosuppression. It was shown that CCR2is required for mobilization of inflammatory (CD11b⁺Ly6C^(hi)) monocytesand accumulation in lung allographs using a mouse model of lungtransplantation²⁹. We used ⁶⁴Cu-DOTA-ECL1i to image this biology in themouse transplant model. Transplantation of a wild type left lung intowild type mice resulted in intense signal in the left lung, consistentwith CCR2-associated reperfusion injury (see e.g., FIG. 6A, red circleleft). Whereas, a wild type lung transplanted into a CCR2^(−/−) mouseshows minimal PET signal, suggesting abrogation of the reperfusioninjury (see e.g., FIG. 6A, left) and that ⁶⁴Cu-DOTA-ECL1i is specificfor the detection of CCR2⁺ inflammation in this model.

Summary of Data.

We demonstrate that: (1) peptide ECL1i is specific for CCR2 in vitro;(2) In an acute LPS lung injury model, ⁶⁴Cu-DOTA-ECL1i PET imaging issensitive to low dose injury, and specific, since it does not signal inCCR2^(−/−) mice after LPS or acute reperfusion injury; a marked⁶⁴Cu-DOTA-ECL1i lung signal is extinguished by CCR2 antibody depletion.Moreover, in Example 4, we show data that ECL1i binds immune cells fromhuman lung, ex vivo (see e.g., FIG. 16).

Example 4: Characterize the Sensitivity, Specificity and Stability of64Cu-DOTA-ECL1i to Detect CCR2 in Mice

Example 4.A. assesses ⁶⁴Cu-DOTA-ECL1i sensitivity to detect CCR2 inmouse models of lung injury. Example 4.B. assesses ⁶⁴Cu-DOTA-ECL1ispecificity to detect CCR2 in mouse models of lung injury. Example 4.C.assesses CCR2 probe stability in target organs in an acute lung injury.

A. Rationale and Experimental Design.

Here, the relationship between the ⁶⁴Cu-DOTA-ECL1i PET signal and theCCR2⁺ cell burden in the lung will be determined by detection using flowcytometry. We will define a true positive (sensitivity)⁹⁰ as thepresence of a PET signal when the burden of CCR2⁺ cells is above a“normal” baseline (naïve mice). Our data suggests that ⁶⁴Cu-DOTA-ECL1iPET can detect differences in naïve and inflammation induced by LPSdoses that differed 5-fold (low vs. intermediate, FIG. 15D), but we havenot quantified CCR2⁺ cells. We will use acute and chronic lunginflammation models. Intratracheal LPS will serve as a reductionistmodel of acute injury, as there is no ideal mouse model of ARDS⁸³.Paramyxovirus (Sendai) infection will serve as the chronic model. Thisis not intended as a model of COPD or asthma, despite shared features ofchronic airway inflammation and mucous cell hyperplasia⁸⁷⁻⁸⁹. Theexperimental design will utilize the CCR2^(RFP) reporter mice (C57BL/6,from D. Kreisel) to facilitate detection of CCR2⁺ cells⁹¹ and avoidunderestimating CCR2⁺ cells after transient receptor endocytosis⁹².These mice are heterozygous for CCR2 and have intact CCR2 responses⁹¹.We generated ECL1i-647 (Alexa Fluor 647) for co-detection of CCR2-RFPand to minimize autofluorescence in flow cytometry (see e.g., FIG. 16D,below). Image analysis will be optimized for lung volume and density byDrs. Chen and Shoghi as described^(72,77,78,93,94). The reproducibilityof ⁶⁴Cu-DOTA-ECL1i imaging in a single animal will be tested in theParamyxovirus model using next-day studies. To visualize ECL1i-647 CCR2⁺cell localization and interactions, we will use 2-photon microscopy,performed by M. Miller (see letter) and D. Kreisel, who have previouslyimaged CCR2^(RFP/+) mice^(29,95).

A. Experimental Procedures (TABLE 3).

Shared control studies in examples 4.A. and 4.B. will minimize thenumber of PET/CT scans. For the acute lung injury studies, a naïve mousewill be studied and LPS dose titrated. Paramyxovirus will be providedwith UV inactivated virus as a control⁸⁷⁻⁸⁹. Histology and geneexpression studies will confirm inflammatory burden. Time points forevaluation are based on prior publications^(25,85,89,96-98). Flowcytometry will use standard surface markers to identify cell types, withexpertise provided by immunologists Kreisel and Byers. PET images willread blind to the condition. For examples 4.A. and 4.B., at least 3-5mice will be studied at each time point, means will be compared byt-test and ANOVA or with non-parametric testing as required. Thepercentage and type of CCR2⁺/CCR2⁻ cells and PET signal intensity willbe analyzed by regression.

TABLE 3 Example 4A Experimental Design: ECL1i tracer sensitivity studiesMouse Time- Strain Injury Model Tracer Assay points Example 4A: GoalsWild type Acute: ⁶⁴Cu-ECL1i; PET, flow 2, 8, Relationship between andnaive vs. Fluor-ECL1i cytometry, 24, 48, PET signal and lungCCR2^(RFP/+) PBS vs. LPS histology 144 h CCR2+ cell burden in acute lunginjury Wild type Chronic: ⁶⁴Cu-ECL1i; PET, flow 5, 12, Relationshipbetween and inactivated Fluor-ECL1i cytometry, 21, PET signal and lungCCR2^(RFP/+) SeV vs. histology 49 d CCR2+ cell burden in SeV chroniclung injury, reproducibility of PET CCR2^(RFP/+) naive vs. Fluor-ECL1i2-Photon 2, 24 h Localization of LPS microscopy CCR2+/ECL1i+ cells

B. Rationale and Experimental Design.

Example 4.B. will determine the specificity of the ⁶⁴Cu-DOTA-ECL1i PETsignal to detect a CCR2⁺ cell burden in the lung. Our data suggests thatthe ECL1i peptide specifically binds to CCR2 and we see an absence of⁶⁴Cu-DOTA-ECL1i PET signal in lungs of CCR2^(−/−) mice subjected to LPSor reperfusion injury (see e.g., FIG. 15C, FIG. 17A). We propose threeadditional studies. First, we will test a scrambled ECL1i sequence inthe radiolabeled and optical tracers (FKLTLCG; tested by C. Combadierein vitro, not shown). Second, to monitor CCR2 molecular therapy we willstudy CCR2 antagonist our acute and chronic injury models, usingRS504393 (Tocris), based efficacy in LPS lung injury and othermodels^(26,99,100). Third, since monocytes are often a dominantCCR2-expressing cell type and modulators of acute and chronic immuneresponses, we will determine the effect of monocyte depletion onimaging. We will use the MaFIA (macrophage Fas-induced apoptosis)transgenic mouse system¹⁰¹ based on the Kreisel lab experience (data notshown) and reports of monocyte depletion in mouse lung disease and othermodels^(34,101). The transgene contains a Fas gene domain and eGFPreporter gene, driven by the colony stimulating factor promoter andactivated by the drug AP20187. To image monocyte depletion, the LPS andlung transplant reperfusion injury models will be used, which have knownmonocyte responses^(25,29,85).

B. Experimental Procedures (TABLE 4).

The magnitude of the ⁶⁴Cu-DOTA-ECL1i/PET signal after the delivery ofintratracheal recombinant mouse CCL2 (50 μg/mouse)²⁵ relative to theburden of CCR2⁺ cells will first be identified by flow cytometry assayand compared in scrambled ECL1i studies. For CCR2 antagonist studies, wedetermine the effect of antagonist pre-treatment (LPS only) andpost-injury treatment (LPS and chronic virus models) on PET signal. Tofollow monocyte depletion the MaFIA mouse will be bred with theCCR2RFP/+ mouse to take advantage of the reporters in each strain.Efficacy of monocyte/macrophage deletion will be confirmed using theGFP-YFP reporter in the transgenic cells and flow cytometry.

TABLE 4 Example 4B Experimental Design: tracer sensitivity studies.Mouse Time- Strain Injury Model Tracer Assay points Example 4B: GoalsCCR2^(RFP/+) Naïve vs. ⁶⁴Cu-ECL1i; PET, flow 8, 24 h Specificity ofCCL2- CCL2 Fluor-ECL1i cytometry, stimulated CCR2 histology activityWild type Naïve vs. ⁶⁴Cu-ECL1i; PET, flow 8, 24 h Effect of scrambledand CCL2, PBS Fluor-ECL1i cytometry, ECL1i peptide on CCR2^(RFP/+) vs.LPS vs. scrambled histology PET vs. CCR2⁺ cells Wild type Acute⁶⁴Cu-ECL1i; PET, flow 2, 8, Effect of pre/post and PBS vs. LPS ±Fluor-ECL1i cytometry, 24, 48, antag. Rx on PET vs. CCR2^(RFP/+)Antagonist histology 144 h CCR2⁺ cells in acute inj. MaFIA × naive vs.⁶⁴Cu-ECL1i; PET, flow 2, 24 h Effect of monocyte CCR2^(RFP/+) LPS andFluor-ECL1i cytometry, depletion on PET vs. Lung Tx histology CCR2⁺cells

C. Rationale, Experimental Design and Procedure.

We have demonstrated that the ⁶⁴Cu-DOTA-ECL1i compound is stable for 1 hafter incubation in mouse serum at 37° C. and in vivo in blood and lungafter injected into a naïve mouse. However, it is possible that locallung and systemic inflammation may alter stability. Stability ofCu-DOTA-ECL1i during lung injury will be assessed in the LPS model withanalysis of target organs (lung, liver, kidney and bone marrow) by HPLCand mass spectrometry. The stability of ⁶⁴Cu-DOTA-ECL1i generated usingGMP conditions will be tested in human serum.

Anticipated Results, Potential Problems, and Alternative Approaches.

These experiments will provide the strength of the relationship betweenthe ⁶⁴Cu-DOTA-ECL1i PET signal and the CCR2 cell burden. We expect tofind that the tracer is relatively sensitive and very specific. Oneissue will to be to determine the lowest CCR2+ population detected byPET, and relate that to a CCR2⁺ cell burden. To augment thisdetermination we can measure CCL2 by ELISA. We expect that there will bea “false negative” condition i.e., a likelihood abnormal levels of CCR2⁺cells present when the PET signal is absent. To determine the pathologiceffect of the CCR2 cell burden in this condition, we will examinehistology and cytokine levels. Low PET signal may be due to theinability of the intravenously delivered tracer to access all CCR2⁺cells, including airspaces^(25,26,85). We will test this by BAL usingγ-counting) and with parallel cell flow cytometry as we have donepreviously¹⁰². We expect that the level of CCR2⁺ cells will track withlung inflammation in general, however, the cell type bearing CCR2 willlikely differ between the acute and chronic models.

Evidence that DOTA-ECL1i-647 binds CCR2^(RFP/+) cells or co-localizeswith CCR2 antibodies will provide evidence of tracer specificity. Weexpect that peptide specificity will also be validated by use of ascrambled ECL1i, a CCR2 antagonist and monocyte depletion. Theantagonist study is of interest as a therapy model. Failure to see aneffect with RS504393 will lead trials of alternative antagonists (e.g.,BMS CCR2 28) or CCR2 antibodies^(25,85). We expect that monocytedepletion will nearly abolish PET signals but have chosen only acutemodels that are known to be CCR2+ monocyte dominant. To study chronicinflammation in future studies we could apply use the MaFIA mice in theParamyxovirus model. Difficulties with the MaFIA transgenic mice willlead us to use liposomal clodronate¹⁰³ or diphtheria toxin transgenicmice¹⁰⁴ for monocyte depletion.

Regarding stability, radiotracer degradation in the mouse injury modelsis not anticipated given the reproducible signal in mouse models.Peptide screening for predicted enzyme targets (ExPASy.org) showed rareenzyme cleavage sites (e.g., pepsin). If degradation is observed, wewill generate the predicted products as HPLC standards to identifydegraded forms, then alter target cleavage sites.

For the radiotracers that meet the criteria for success we will assessradiolabeling the peptide with ⁶⁸Ga, a positron emitter with desirablenuclear properties (t_(1/2)=68 min, β⁺% 89%, E_(β+max): 1.92 MeV)produced by commercial available ⁶⁸Ge/⁶⁸Ga generator, to expand thetranslation potential. Imaging with this radiotracer will be compared to⁶⁴Cu-DOTA-ECL1i in the mouse lung injury models. If ultimately humansafety studies are satisfactory ⁶⁸Ga-DOTA-ECL1i and resolution isretained with ⁶⁸Ga, the lower radiation exposure and availability of theradionuclide may allow repeated scans in patients with ongoing symptomsto test therapeutic responses.

Example 5: Assess 64Cu-DOTA-ECL1i Binding to hCCR2 and Quantify CCR2Activity in Human Lung Tissues

Example 5.A. determines the sensitivity, specificity, and stability of⁶⁴Cu-DOTA-ECL1i to bind hCCR2 in cells. Example 5.B. evaluates theability of ECL1i-based probes to detect CCR2 in human lung cells andtissues. Example 5.C. describes a Phase 0/Early Phase 1 trial todetermine ⁶⁴Cu-DOTA-ECL1i safety and dosimetry in healthy volunteers.

A. Rationale and Experimental Design.

The goal of this example is to assess the performance of the ECL1itracer in human cells. The amino acid sequence of the CCR2 in the regionof the extracellular loop 1 used to design ECL1i is >95% conserved inthe mouse and human. Our data using autoradiography suggest that⁶⁴Cu-DOTA-ECL1i binds human CCR2 in tissues (see e.g., FIG. 16C, below).To measure the specific binding of ECL1i to human CCR2 we have produceda human CCR2 expression plasmid to generate a stable cell line in humanHEK293 cells. The approach has been used our group to study ligandbinding for PET radiotracers¹⁰⁵ and will be used to develop an affinitybinding assay for both experimental and post-release quality control(QC) of ⁶⁴Cu-DOTA-ECL1i. As in mice, we will test the specificity usingthe scrambled ECL1i sequence and the stability using HPLC and massspectroscopy to characterize the performance of ⁶⁴Cu-DOTA-ECL1i in humancells.

A. Experimental Procedures.

A HEK293-hCCR2 stable cell line will be validated at the RNA and proteinlevels, compared to a control cell line transfected with an emptyvector. We will generate a set of HEK293 cells expressing additionalhuman chemokine receptors (e.g., CCR1, CCR5, CXCR4) or obtain these fromC. Combadiere¹⁰⁶. For the affinity binding assays ⁶⁴Cu-DOTA-ECL1i (vs.the scrambled version) will be incubated with the cells in a range ofconcentrations in the absence or presence of excess non-radiolabeledCu-DOTA-ECL1i and counted in a L-counter to calculate an IC₅₀. An SOPwill be developed and the assay applied as a QC measure for⁶⁴Cu-DOTA-ECL1i production. Parallel studies will be performed with andECL1i-647. Stability studies of ⁶⁴Cu-DOTA-ECL1i will be performed inhuman serum as described in Example 4C.

B. Rationale and Experimental Design.

The goal of this example is to characterize the binding of⁶⁴Cu-DOTA-ECL1i in normal and diseased lung tissues. Studies will alsovalidate CCR2 in a lung disease population. We will determine thecapacity of ⁶⁴Cu-DOTA-ECL1i and ECL1i-647 to bind hCCR2 usingautoradiography, flow cytometry and two-photon microscopy. We takeadvantage of a substantial, established tissue biorepository previouslyused for COPD studies^(33,87,88,107). We have chosen COPD tissues thisand other reasons: (1) CCL2/CCR2 is present in BAL of subjects withasthma^(34,108,109), but to validate CCR2 expression in interstitialtissues by transbronchial biopsy is potentially risky for thispopulation. (2) Levels of CCL2/CCR2 are also high in PGD post-lungtransplantation, but despite routine bronchoscopy in this group, anunstable status is not ideal for tissue validation studies. (3) ElevatedCCL2/CCR2 in COPD BAL and in tissues resected for lung cancer³⁰⁻³²,suggested we could use a similar approach.

ECL1i Binds hCCR2 in Lungs from Subjects with COPD.

We found that tissues from lungs explanted from subjects with severeCOPD (removed for lung transplantation) have a mean increase in levelsof CCR2 expression, compared to normal donated lungs (“donor”), byimmunostaining (see e.g., FIG. 16A, FIG. 16B) and a similar pattern inRNA samples (not shown). There were three CCR2 phenotypes (high,intermediate, normal), with implications for future therapies.Importantly, autoradiography of lung sections on slides using⁶⁴Cu-DOTA-ECL1i showed marked increased binding in COPD samples comparedto normal donor (see e.g., FIG. 16C). Our tissue bank also includescells digested from lung for flow cytometry studies. In the reportedstudies, we found that ECL1i bound a population of CD45⁺ immune cells inCOPD lung (see e.g., FIG. 16C). We obtain approximately 6-8 explantedlungs per year from COPD subjects. We propose to also examine lungtissue incubated with ECL1i-647 and cells specific antibodies usingtwo-photon microscopy to localize ECL1i.

B. Experimental Procedures.

We will use explanted lung samples available from over 30 uniquesubjects with advanced COPD that underwent lung transplantation and 15tissues from non-COPD subjects donated for transplantation.Immunostaining with CCR2 and standard antibodies will identify thelocation and extent of CCR2⁺ classical monocytes. Parallel samples offlow cytometry using antibodies and DOTA-ECL1i-647 will quantify andcharacterize CCR2-expressing cells (as in FIG. 3C, FIG. 15, FIG. 16D).At least 4 different regions from each lung are banked for analysis toassess for intra-organ variability. Parallel samples will be used for⁶⁴Cu-DOTA-ECL1i autoradiograph and binding then quantified. These willbe correlated with antibody-based data. For 2-photon microscopy, we willincubate fresh human COPD lung tissue with ECL1i-647 and cell-typespecific antibodies to complement binding observe by autoradiographs andimmunostaining¹¹⁰.

Anticipated Results, Potential Problems and Alternative Approaches.

Receptor binding assays rely on adequate levels of cell surface CCR2expression. If levels are low, we will generate and select cell cloneswith high levels and confirm surface binding using CCR2 antibodies.Primary peripheral blood human monocytes have high surface CCR2expression and may serve as an alternative to cell lines forassays^(111,112).

In studies of tissue from COPD subjects, we expect to find a CCR2expression within the interstitial compartment (out of reach ofbronchoscopy or sputum sampling) on multiple cell types. We also predictthat expression will vary among different samples of the same lung,confirming the overall approach that we are taking and highlighting theproblem inherent in sampling. The variation in levels of CCR2 expressionand ⁶⁴Cu-DOTA-ECL1i binding will support the importance of using PETimaging as a diagnostic test. The tissue bank also includes lung samplesfrom a range of COPD (GOLD 0-IV), making it possible to determine if theCCR2 levels are related to disease severity. Moreover, a large number ofsamples from subjects with idiopathic pulmonary fibrosis (IPF) are alsoavailable. Increased numbers of CCR2 cells are reported inIPF^(18,39-41), which may serve as an alternative population for testingthe ECL1i tracer in human tissue.

Example 6: Translate 64Cu-DOTA-ECL1i in a Phase 0/Early Phase 1 Trial

Example 6.A performs animal toxicology and dosimetry studies of⁶⁴Cu-DOTA-ECL1i. Example 6.B. develops chemistry, manufacturing, andcontrols (CMC) and standard operating procedures (SOPs) for⁶⁴Cu-DOTA-ECL1i production, and submit an elND application.

A. Rationale and Experimental Design.

This example will generate toxicology and dosimetry studies required foran elND application. We will conduct the toxicology study in mice aspreviously performed. Dosimetry will be performed using standardprotocols and analyzed by our investigator with specific expertise inthis area, R. Laforest⁷⁰⁻⁷⁴. Our group has extensive experience inperforming such studies and a core with dedicated personnel andequipment to perform dosimetry studies in a routine fashion.

A. Experimental Procedures.

Rodent toxicity studies. For toxicology studies, non-radioactive copperlabeled Cu-DOTA-ECL1i will be produced following the protocol for ⁶⁴Culabeled production. A single i.v. injection of 100-times the expectedhuman dose per body surface area will be used. Mice, 8-10 weeks of ageof both sexes will evaluated over 14 days to determine the systemictoxicity and organ toxicity by gross necropsy and histology, hematology,blood for clinical pathology analysis, urine analysis and weight lossanalysis. Detailed analysis of the data will be provided for an elNDapplication.

Animal Dosimetry and Estimation for Humans.

Animal biodistribution will be designed according to our previousexperience using ⁶⁴Cu-labeled agents with fast renal clearance andperformed to determine the percent injected dose per gram of tissue (%ID/g) in major organs (>20) at multiple time points after⁶⁴Cu-DOTA-ECL1i injection^(74, 75). The results will be scaled to humanby the relative organ weight method¹¹². Time-activity curves will becreated and radiotracer uptake or clearance functions will be analyzed.Integration of those functions will provide organ residence times. Anadditional group of animals will be kept in metabolic cages to determinethe excretion.

Human Radiation Dose Estimates.

This will be calculated from the mouse dosimetry data, residence timesand for the standard human male model using OLINDA-EXE (version 1.1) asdescribed¹¹³.

B. Rationale and Experimental Design.

The purpose of this example is to develop protocols for production ofthe radiotracer and complete the chemistry manufacturing and controls(CMC) and standard operating procedures (SOPs) for an elND applicationfor human use. We will take advantage of our extensive experience inthis regard and the use of our ISO 7 GMP facility, nuclear pharmacy andcyclotron production facility (see Resources). Members of the Liu labwill work closely with investigator S. Schwarz to develop new SOPs andfor scale-up production to prepare elND documents, together withtoxicology and dosimetry data from Example 6.A.

B. Experimental Procedures.

The production of DOTA-ECL1i following SOPs under cGMP conditions willbe scaled up and full QC characterization will be performed. Stabilityof DOTA-ECL1i as raw material will be monitored to provide guidanceabout the expiration of the probe. Validation runs of ⁶⁴Cu labeling withfull process and quality control will be performed in the cGMP facilityfollowing batch production record and SOPs for elND submission.

C. Rationale and Experimental Design.

The goals of this example are to determine human safety, biodistributionand radiation dosimetry of ⁶⁴Cu-DOTA-ECL1i. This study will be a singlecenter, open-label baseline controlled imaging study. Plannedrecruitment will be for 18 subjects with a final goal to study 12 fordosimetry determination based on our prior studies^(70,72-74). We willinclude non-smokers and cigarette smokers with normal pulmonary function(spirometry) to determine if systemic inflammation that is well known insmokers^(113,114) alters dosimetry^(115,116). Potential studypopulations with acute and chronic lung disease are often cigarettesmokers.

C. Experimental Procedures.

Regulatory Approval:

The second 6 months of year 2 will be devoted obtaining FDA approval viathe elND mechanism and IRB and RDRC approvals at Washington University.

Research Subjects.

The safety and dosimetry of ⁶⁴Cu-DOTA-ECL1i will be tested in 12 healthyvolunteer subjects using a single intravenous dosage. Non-smoking (6)and tobacco smoking, with normal lung spirometry (6) will be included. Atotal of 18 subjects will be recruited with an expect non-completionrate of 20%. Our research coordinator will recruit subjects withassistance from the Volunteers for Health Program at the School ofMedicine (https://vfh.wustl.edu/), an approach that has been successfulover the past 15 years. Subjects will be contacted by telephone andinterviewed for suitability and inclusion/exclusion criteria. These willbe as follows: Inclusion: Healthy man or woman, any race or ethnicity,age 21-45 years old; screening FEV1 and FVC >90% of predicted insmokers; capable of lying still, supine within the PET/CT scanner for˜1.5 hours and following instructions for breathing protocol during theCT portion; a BMI <35 and able and willing to give informed consent.Exclusion: Pregnancy (confirmed by serum hCG test); lactation; activemenstruation; active symptoms or history of cardiopulmonary, diabetes,hepatic or renal disease; current use of prescription medications;history of illicit drug use within the past year; enrollment in anotherresearch study of an investigational drug.

General protocol (see e.g., FIG. 18). Once recruited, consent forms willbe provided the subjects for review at least 24 h prior to the studies.Subjects will be met in the Center for Clinical Imagining and Research(CCIR), within Barnes-Jewish Hospital. Consent will be obtained and ahistory and physical exam performed by a physician investigator. Forsafety analysis subjects will undergo vital sign measurement and testingas in FIG. 18. Results of laboratory tests (complete blood count,comprehensive metabolic panel, and urinalysis, plus pregnancy test forwomen and spirometry for cigarette smokers) will be obtained prior toinjection. Subjects with abnormal tests will be withdrawn andcompensated.

⁶⁴Cu-DOTA-ECL1i Injection and Imaging Protocol.

The radiotracer will be prepared in the GMP facility as described inExample 6B. A pre-dosimetry dose estimate is 0.7 μg/kg. A peripheralintravenous catheter will be place. Vital signs will be obtainedpre-injection (within 30 min prior to injection of ⁶⁴Cu-DOTA-ECL1i),10-15 min post injection; and at completion of each imaging cycle.Volunteers will be undergo whole-body PET/CT imaging at a paired timesbetween 0 and 50 h post injection to calculate human dosimetry based onbiodistribution of this ⁶⁴Cu-based, peptide containing radiotracer. Fourgroups (G1-4) of 3 patients each will sample the 50 h post injectionperiod with 2 scans each: G1 0-3 h and 4-6 h; G2 0-3 h and 22-28 h; G34-6 h and 22-28 h G4 1-3 h and 46-50 h. Although allergic or otherimmediate adverse reactions are not anticipated, subjects will bemonitored for at least one-hour post injection in an area whereemergency equipment is available. The study will terminate if subjectsexperience any sign of toxicity or tracer-associated discomfort.Subjects will remain in the imaging unit for serial exams or return forsubsequent scan, post-scan testing, and compensation. Subject statuswill be determined in a follow-up phone call at one day after the lastscan.

PET/CT Image Acquisition.

PET/CT acquisition will include a CT scan for attenuation correctionwith the patient supine after intravenous administration of⁶⁴Cu-DOTA-ECL1i. The CT will consist of a 10-20 second topogram fordetermining correct anatomical positioning followed by a spiral CT at 50mAs (or Caredose calculated dose if less than 50 mAs) and 120 kVp.Average CT scan time is 15-30 seconds to acquire a 5-mm-slices.Immediately after the attenuation CT scan, emission images from the topof the skull through the upper thighs will be obtained (1-10 min per bedposition).

Image Analysis, Calculation of Biodistribution and Radiation Dosimetry:

Organ activity concentration will be measured on the most visible organson the PET images. The average activity concentration measured in organswill be converted to percent injected dose¹¹⁷. Time activity curvescombining the percent-injected dose for all subjects will then becreated. Activity residence times for each observed organ will then becalculated and corrected for radioactive decay. The urinary bladderexcretion is calculated using an established model¹¹⁸ Radiation dosesestimates will then be calculated using the OLINDA/EXM (version 1.1)¹¹³in the standard human male model.

Anticipated Results, Potential Problems and Alternative Approaches.

Pre-clinical studies are expected to have minimal adverse effects,although toxicity is unpredictable. If significant toxicity is observedin mice, we will attempt to isolate the source of toxicity. One sourceof peptide toxicity may be charge. Change will be reduced byN-acetylation and C-amidation of the ECL1i peptide, (which will notaffect DOTA conjugation). Also, acetylation may reduce toxicity as acloser mimic of native protein, since 85% of human proteins areacetylated¹¹⁷.

Because the manufacturing methods are well established, we do not expectproblems with scale up production and radiolabeling. Based on priorexperience, we anticipate an approximate 10% failure rate in ⁶⁴CU or⁶⁴Cu-DOTA-ECL1i production. If this is the case, we have includedreimbursement funds for recruited subjects who are unable to completethe study due to this technical issue.

We will design our human dosimetry protocol based on mouse dosimetry. Weexpect that the kidney or bladder wall will be the radiationdose-limiting organ. Given we will be administering <100 μg of product,in accordance with elND guidelines, we do not expect to observe serioustoxic effects but are aware of potential CCR2 antagonism of ECL1i.Consequently, subjects will be monitored for at least one-hour postinjection in an area where emergency equipment is available. In theevent of higher than expected radiation dose to the kidneys or bladderwall, dose reduction strategy in humans will include appropriate patienthydration and regular bladder voiding. We will immediately halt thestudy if there are any significantly symptoms or laboratoryabnormalities and notify the FDA and IRB.

Future directions: Proposed Phase 1 ⁶⁴Cu-DOTA-ECL1i safety studies insubjects with lung inflammation.

Once our early Phase 1 trial confirms safety in healthy volunteers, wewill evaluate the performance of ⁶⁴Cu-DOTA-ECL1i in target populationsfor later Phase 1 studies, where knowing the inflammatory burden willaid in clinical decision making. We are considering two potential studypopulations for safety assessment. First, ⁶⁴Cu-DOTA-ECL1i PET imagingfollowing segmental endotoxin challenge delivered by bronchoscopy⁷⁶⁻⁷⁸.Post-endotoxin bronchoscopy and BAL permits potential assessment ofCCR2⁺ cells in the airspace. A second consideration are individuals withsevere COPD awaiting lung transplantation, who offer the potential toassay the CCR2⁺ cell burden in explanted lungs for comparison to theintensity of PET image. Subjects with COPD undergoing surgery for lungnodules may also provide tissue for assessing CCR2⁺ cell status, thoughCCR2⁺ macrophage may infiltrate the tumor¹¹⁹. The unstable nature ofpatients with respiratory failure groups such as those with ARDS andresolving vs. persistent inflammation or the post lung transplantpatient with PGD may be too high risk for phase 1 studies.

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What is claimed is:
 1. An imaging agent comprising: a CCR2 bindingpeptide; a radiolabel; and a nanoparticle, a chelator, or a linker. 2.The imaging agent of claim 1, further comprising a linker.
 3. Theimaging agent of claim 1, wherein the CCR2 binding peptide comprises:(i) a linear ECL1 peptide or a cyclized ECL1i peptide; (ii) an aminoacid sequence, Thr-Phe-Leu-Lys (SEQ ID NO: 17); (iii) an amino acidsequence, SEQ ID NO: 17, comprising one or more chemical modificationsthat confers resistance to proteolysis; (iv) an amino acid sequence, SEQID NO: 17, comprising one or more conservative substitutions; (v) anamino acid sequence, Thr-Phe-Leu-Lys-Cys (SEQ ID NO: 1); (vi) an aminoacid sequence, SEQ ID NO: 1 comprising one or more chemicalmodifications that confer resistance to proteolysis; (vii) an amino acidsequence, SEQ ID NO: 1 comprising one or more conservativesubstitutions; (viii) an amino acid sequence, X1-TFLKC-X2 (SEQ ID NO:2), wherein X1 is absent, is glycine, or represents an amino acidsequence selected from the group consisting of AG, LG, YLG, and HYLG;and X2 independently is absent, is methionine, or represents an aminoacid sequence selected from the group consisting of MA, MAN, MANG,MANGF, MANGFV, MANGFVW, MANGFVWE, and MANGFVWEN; (ix) an amino acidsequence, SEQ ID NO: 2 comprising one or more chemical modificationsthat confer resistance to proteolysis; (x) an amino acid sequence, SEQID NO: 2 comprising one or more conservative substitutions; (xi) anamino acid sequence, X1-TFLK-X3 (SEQ ID NO: 18), wherein X1 is absent,is glycine, or represents an amino acid sequence selected from the groupconsisting of AG, LG, YLG, and HYLG; and X3 independently is absent oris alanine; (xii) an amino acid sequence, SEQ ID NO: 18, comprising oneor more chemical modifications that confer resistance to proteolysis;and (xiii) an amino acid sequence, SEQ ID NO: 18, comprising one or moreconservative substitutions.
 4. The imaging agent of claim 1, wherein theCCR2 binding peptide comprises amino acids and all or a portion of theamino acids are in L configuration or in D configuration; the imagingagent is stored in a physiological pH, optionally, at about pH of 7.4;the CCR2 binding peptide is covalently linked to the nanoparticle; orthe CCR2 binding peptide is no more than 18 amino acids in length. 5.The imaging agent of claim 1, wherein the CCR2 binding peptide isselected from the group consisting of: LGTFLKC (SEQ ID NO: 3);HYLGTFLKCMA (SEQ ID NO: 4); LGTFLKCMA (SEQ ID NO: 5); HYLGTFLKC (SEQ IDNO: 6); GTFLKCMANGF (SEQ ID NO: 7); TFLKCMANGFV (SEQ ID NO: 8);HYLGTFLKCMANGFVWEN (SEQ ID NO: 9); LGTFLK (SEQ ID NO: 19); AGTFLKC (SEQID NO: 20); LGTFLKA (SEQ ID NO: 21); GTFLK (SEQ ID NO: 22); AGTFLKA (SEQID NO: 23); a sequence deriving from any of SEQ ID NO: 3 to 10, or 19 to23 by one or more chemical modifications that confer resistance toproteolysis; and a sequence deriving from any of SEQ ID NO: 3 to 9 or 19to 23 by one or more conservative substitutions.
 6. The imaging agent ofclaim 5, wherein the CCR2 binding peptide consists of LGTFLKC (SEQ IDNO: 3).
 7. The imaging agent of claim 1, wherein (i) the radiolabelcomprises ²H (D or deuterium), ³H (T or tritium), ¹¹C, ¹³C, ¹⁴C, ⁶⁴Cu,⁶⁷Cu, ¹⁷⁷Lu, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ⁸⁹Sr, ³⁵S, ¹⁵³Sm, ³⁶Cl, ⁸²Br,⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Tl ^(99m)Tc, ⁹⁰Y, or ⁸⁹Zr; (ii) the radiolabelcomprises oxygen-15 water, nitrogen-13 ammonia, [⁸²Rb] rubidium-82chloride, [¹¹C], [¹¹C] 25B-NBOMe, [¹⁸F] Altanserin, [¹¹C] Carfentanil,[¹¹C] DASB, [¹¹C] DTBZ, [¹⁸F]Fluoropropyl-DTBZ, [¹¹C] ME@HAPTHI, [¹⁸F]Fallypride, [¹⁸F] Florbetaben, [¹⁸F] Flubatine, [¹⁸F] Fluspidine, [¹⁸F]Florbetapir, [¹⁸F] or [¹¹C] Flumazenil, [¹⁸F] Flutemetamol, [¹⁸F]Fluorodopa, [¹⁸F] Desmethoxyfallypride, [¹⁸F] Mefway, [¹⁸F] MPPF, [¹⁸F]Nifene, Pittsburgh compound B, [¹¹C] Raclopride, [¹⁸F] Setoperone, [¹⁸F]or [¹¹C] N-Methylspiperone, [¹¹C] Verapamil, [¹¹C] Martinostat,Fludeoxyglucose (¹⁸F)(FDG)-glucose analogue, [¹¹C] Acetate, [¹¹C]Methionine, [¹¹C] Choline, [¹⁸F] Fluciclovine, [¹⁸F] Fluorocholine,[¹⁸F] FET, [¹⁸F] FMISO, [¹⁸F] 3′-fluoro-3′-deoxythymidine, [⁶⁸Ga]DOTA-pseudopeptides, [⁶⁸Ga] PSMA, or [¹⁸F] Fluorodeoxysorbitol (FDS);(iii) the chelator comprises NHS-MAG₃, MAG₃, DTPA, 3p-C-NE3TA,3p-C-NOTA, 3p-C-DE4TA, ATSM, tetraazamacrocyclic ligands (e.g., DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTA-NHS,pSCN-Bn-DOTA, pNH₂-Bn-DOTA, TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid,TETA-octreotide (OC)), hexaazamacrobicyclic cage-type ligands (e.g.,Sarcophogine chelators), cross-bridged tetraamine ligands (e.g., CB-TE2A(4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane)),6-Hydrazinopridine-3-carboxylic acid (Hynic), NHS-Hynic,2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA); or (iv) the nanoparticle comprises ananocluster or nanostructure; organic, inorganic, or lipidnanostructures; (v) the nanoparticle comprises iron oxide, gold, goldnanoclusters (AuNC), gold nanorods (AuNR), copper (Cu), quantum dots,carbon nanotubes, carbon nanohorn, gadolinium (Gd), dendrimers,dendrons, polyelectrolyte complex (PEC) nanoparticles, calcium phosphatenanoparticles, perfluorocarbon nanoparticles (PFCNPs), lipid-basednanoparticles, liposomes, or micelles; or (vi) the linker comprises achemical or physical bond; PEG, TA-PEG-Maleimide, TA-PEG-OMe, TA-PEG, anisothiocyanate group, a carboxylic acid or carboxylate groups, adendrimer, a dendron, Fmoc-protected-2,3-diaminopropanoic acid, ascorbicacid, a silane linker, minopropyltrimethoxysilane (APTMS), dopamine, 2thiol groups, 2 primary amines, a carboxylic acid and primary amine,maleimide and thiol, hydrazide and aldehyde, or a primary amine andaldehyde, an amide, a thioether, a disulfide, an acetyl-hydrazone group,a polycyclic group, a click chemistry (CC) group.
 8. The imaging agentof claim 1, wherein (i) the chelator is conjugated to the CCR2 bindingpeptide and the chelator is radiolabeled; or (ii) the CCR2 bindingpeptide is conjugated to a nanoparticle.
 9. The imaging agent of claim8, wherein the radiolabel is ⁶⁴Cu.
 10. The imaging agent of claim 8,wherein the CCR2 binding peptide is conjugated to a nanoparticlecomprising a gold nanocluster.
 11. The imaging agent of claim 10,wherein the gold nanocluster is loaded with a radiolabel.
 12. Theimaging agent of claim 1, wherein the chelator comprises atetraazamacrocyclic ligand; DOTA; TETA; or2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid (Maleimido-mono-amide-DOTA).
 13. The imaging agent of claim 1,wherein the chelator is conjugated to a cysteine residue of the CCR2binding peptide.
 14. The imaging agent of claim 1, wherein the imagingagent comprises: (i) a ⁶⁴Cu-DOTA-ECL1i PET or SPECT imaging agent; (ii)an ECL1i peptide conjugated to a gold nanocluster, wherein the goldnanocluster is loaded with ⁶⁴Cu; (iii) ⁶⁴Cu-DOTA-ECL1i; (iv)⁶⁴CuAuNCs-ECL1i; (v) a multivalent imaging agent (optionally,⁶⁴CuAuNCs-ECL1i), exhibiting extended pharmacokinetics for long-termCCR2 receptor detection and targeted theranostics; or (vi) a monovalent(optionally, ⁶⁴Cu-DOTA-ECL1i), exhibiting fast pharmacokinetics forefficiently for rapid or serial imaging of CCR2 receptors.
 15. Theimaging agent of claim 1, wherein the imaging agent is a PET imagingagent; is a SPECT imaging agent; targets CCR2 receptors; detects CCR2receptor up-regulation; or detects elevated CCR2 expression.
 16. Amethod of detecting a CCR2 receptor comprising: administering to asubject an imaging agent of claim 1; and detecting the imaging agent.17. The method of claim 16, wherein detecting the CCR2 receptorcomprises positron emission tomography (PET) imaging, and single photonemission computed tomography (SPECT) imaging, mass spectrometry, gammaimaging, magnetic resonance imaging (MRI), magnetic resonancespectroscopy, fluorescence spectroscopy, CT, ultrasound, or X-ray. 18.The method of claim 17, wherein the method detects a CCR2 associateddisease, disorder, or condition is selected from the group consistingof: (i) atherosclerosis; abdominal aortic aneurysm; acquired metabolicdisease; acute cystitis; acute lung injury; acute proliferativeglomerulonephritis; acute or chronic sinusitis; age-related maculardegeneration; alcoholic hepatitis; allergic asthma; allergicconjunctivitis; allergic rhinitis; alveolitis; angiostenosis;anthracosis; ariboflavinosis; arteriosclerosis; artery disease;arthritis; asthma; atherogenesis; atheroma; atherosclerosis; atopicdermatitis; autoimmune disease; autoinflammation; bacterial infection;bacteriuria; bladder cancer; bone cancer; bone inflammation disease;brain trauma; breast cancer; bronchiolitis; bronchiolitis obliteranssyndrome; cancer; cardiac infarction; cardiovascular disease; carotidartery disease; CCR2 associated neurological disorders; Cd3zetadeficiency; central nervous system disease; cerebral aneurysms; cervicalcancer; chagas disease; chorioamnionitis; chronic heart failure; chroniclung disease; chronic lymphocytic leukemia; chronic myelocytic leukemia;chronic obstructive pulmonary disease (COPD); chronic respiratory viralinfection; chronic urticaria; colitis; colon cancer; complex regionalpain syndrome; coronary artery aneurysm; crescentic glomerulonephritis;Crohn's Disease; cystitis; cytomegalovirus retinitis; degeneration ofmacula and posterior pole; demyelinating disease; dengue shock syndrome;denture stomatitis; dermatosis syndrome; diabetes; diabetes mellitus,noninsulin-dependent; diabetic angiopathy; diabetic complications;diabetic macular edema; diabetic microangiopathy; diabetic nephropathy;diabetic retinitis; diabetic retinopathy; diastolic cardiomyopathies;encephalitis; endocervicitis; endometrial stromal sarcoma;endometriosis; Erdheim-Chester disease; extrapulmonary tuberculosis;extrinsic cardiomyopathy; eye disease; fibroid lung; fungal pneumonia;gingivitis; glomerulonephritis; gum disease; Hamman-Rich syndrome; headand neck cancer, herpes simplex virus keratitis; HIV-1; Hodgkin'sdisease; hyperhomocysteinemia; idiopathic anterior uveitis; idiopathicinterstitial pneumonia; idiopathic pulmonary fibrosis; inflammationafter cataract surgery; inflammatory bowel diseases; inflammatorydisease; influenza; interstitial lung disease; invasive staphyloccocia;ischemia of lower members of the heart; ischemia-reperfusion injury;Israeli tick typhus; Kawasaki disease; keratitis; kidney disease;leptospirosis; limb ischemia; lipid pneumonia; lipodystrophy; lipoidnephrosis; lung cancer; lung disease; lung injury; lung transplantation;macular degeneration, age-related, 1; macular holes; malaria; malignantmyeloma; mast-cell leukemia; meningitis; mesangial proliferativeglomerulonephritis; metabolic disease; microvascular complications ofdiabetes 1; monocytic leukemia; multiple myeloma; multiple sclerosis;mycobacterium tuberculosis; myocardial infarction; myocarditis;necrosis; neovascular inflammatory disease; nephritis; nephrosclerosis;neural tube defects; neuritis; neuroinflammation; nonspecificinterstitial pneumonia; obesity; ophthalmic disorder; organ allograftrejection; overnutrition; pain; pain from a sciatic nerve; papillaryconjunctivitis; pelvic inflammatory disease; periodonitis; periodontaldiseases; periodontitis; peripheral artery disease; peripheral pain;peritonitis; pleural tuberculosis; pleurisy; pneumoconiosis; pneumonia;post-thrombotic syndrome; primary graft dysfunction (PGD) (a reperfusioninjury after transplant); proliferative glomerulonephritis; prostatecancer; psoriasis; psoriatic arthritis; pulmonary alveolar proteinosis;pulmonary fibrosis; pulmonary sarcoidosis; purulent labyrinthitis;pyelonephritis; radiculopathy; renal fibrosis; renal insufficiency;reperfusion disorders; respiratory system disease; restenosis; retinaldegeneration; retinal vascular occlusion; retinal vasculitis; retinalvein occlusion; rheumatoid arthritis; rhinoscleroma; sarcoidosis;sarcoidosis 1; scleritis; secondary progressive multiple sclerosis;severe acute respiratory syndrome; silicosis; solid tumor; stachybotryschartarum; stomach cancer; stromal keratitis; systemic lupuserythematosus; transient cerebral ischemia; transplant arteriosclerosis;trypanosomiasis; tuberculosis; tuberculous meningitis; type II diabetes;ulcerative colitis; ureteral disease; urinary system disease; urinarytract obstruction; uveitis; vangl1-related neural tube defect; vasculardisease; vascular permeability and attraction of immune cells duringmetastasis; vasculitis; verruciform xanthoma of skin; viral infection;viral encephalitis; viral meningitis; vitreoretinopathy; orxanthogranulomatous pyelonephritis; (ii) acute lung injury;inflammation; primary graft dysfunction (PGD); asthma; pulmonaryfibrosis; COPD; atherosclerosis; lung transplant; lung injury; COPD;atherosclerosis; cancer; prostate cancer; organ transplant; metabolicdisease, type II diabetes; multiple sclerosis; rheumatoid arthritis;pain; pulmonary fibrosis; or reperfusion in a lung transplant; (iii)inflammation associated with a CCR2 associated disease, disorder, orcondition; and (iv) inflammation associated with lung injury, grafttransplantation, atherosclerosis, tumor cells, or cancer.
 19. The methodof claim 16, further comprising: (i) evaluating or monitoring a CCR2associated disease, disorder, or condition; or (ii) administering a CCR2antagonist to a subject.
 20. A pharmaceutical composition comprising theimaging agent of claim 1.